U.S. patent number 9,890,199 [Application Number 15/103,173] was granted by the patent office on 2018-02-13 for staphylococcus aureus spa5 mutant, composition comprising mutant and preparation method and use thereof.
This patent grant is currently assigned to Olymvax Biopharmaceuticals Inc., Third Military Medical University, PLA. The grantee listed for this patent is OLYMVAX BIOPHARMACEUTICALS INC., THIRD MILITARY MEDICAL UNIVERSITY, PLA. Invention is credited to Changzhi Cai, Yandong Dong, Shaowen Fan, Qiang Feng, Haiming Jing, Lu Lu, Yi Wu, Hao Zeng, Jinyong Zhang, Quanming Zou.
United States Patent |
9,890,199 |
Zou , et al. |
February 13, 2018 |
Staphylococcus aureus SpA5 mutant, composition comprising mutant
and preparation method and use thereof
Abstract
Provided is staphylococcus protein A expressed by a mutational
Staphylococcus aureus and its coding sequence, as well as a vector,
host bacteria, composition or kit which contains the coding
sequence of the mutational protein. Also provided is the use of the
mutational protein and the composition thereof in the preparation
of vaccines, therapeutic antibodies, diagnostic kits and the like,
and for the prevention, treatment and detection of infections by
Staphylococcus aureus. Also provided are methods for producing,
fermenting and purifying the mutational protein.
Inventors: |
Zou; Quanming (Chongqing,
CN), Zeng; Hao (Chongqing, CN), Fan;
Shaowen (Chongqing, CN), Lu; Lu (Chongqing,
CN), Feng; Qiang (Chongqing, CN), Zhang;
Jinyong (Chongqing, CN), Jing; Haiming
(Chongqing, CN), Dong; Yandong (Chongqing,
CN), Wu; Yi (Chongqing, CN), Cai;
Changzhi (Chongqing, CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
OLYMVAX BIOPHARMACEUTICALS INC.
THIRD MILITARY MEDICAL UNIVERSITY, PLA |
Chengdu, Sichuan
Chongqing |
N/A
N/A |
CN
CN |
|
|
Assignee: |
Olymvax Biopharmaceuticals Inc.
(Chengdu, CN)
Third Military Medical University, PLA (Chongqing,
CN)
|
Family
ID: |
53370436 |
Appl.
No.: |
15/103,173 |
Filed: |
December 9, 2013 |
PCT
Filed: |
December 09, 2013 |
PCT No.: |
PCT/CN2013/088880 |
371(c)(1),(2),(4) Date: |
June 09, 2016 |
PCT
Pub. No.: |
WO2015/085463 |
PCT
Pub. Date: |
June 18, 2015 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20160304566 A1 |
Oct 20, 2016 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K
39/085 (20130101); C07K 16/12 (20130101); A61K
39/40 (20130101); G01N 33/56938 (20130101); C07K
16/1271 (20130101); C12N 15/70 (20130101); A61P
31/04 (20180101); C07K 14/31 (20130101); A61K
2039/55505 (20130101); G01N 2469/20 (20130101); A61K
2039/575 (20130101); G01N 2333/31 (20130101); C07K
2319/00 (20130101); G01N 2800/26 (20130101) |
Current International
Class: |
C07K
14/31 (20060101); C12N 15/70 (20060101); C07K
16/12 (20060101); G01N 33/569 (20060101); A61K
39/40 (20060101); A61K 39/085 (20060101); A61K
39/00 (20060101) |
Field of
Search: |
;424/130.1,150.1,184.1,185.1,234.1,237.1 |
Foreign Patent Documents
|
|
|
|
|
|
|
0284368 |
|
Sep 1988 |
|
EP |
|
2011005341 |
|
Jan 2011 |
|
WO |
|
2012003474 |
|
Jan 2012 |
|
WO |
|
Other References
Kim et al., Nontoxigenic Protein A Vaccine for
Methicillin-resistant Staphylococcus aureus Infections in Mice,
Journal of Experimental Medicine, Aug. 30, 2010 (Aug. 30, 2010),
No. 9, vol. 207, ISSN: 0022-1007, pp. 1863-1870, see the entire
text. cited by examiner .
Extended European Search Report from European Application No.
13899076.7 dated Jun. 21, 2017. cited by applicant .
Falugi et al., "Role of Protein A in the Evasion of Host Adaptive
Immune Responses by Staphylococcus aureus," mbio.asm.org, vol. 4,
Issue 5, Sep./Oct. 2013, 9 pages. cited by applicant .
Kobayashi et al., "Staphylococcus aureus Protein A Promotes Immune
Suppression," mbio.asm.org, vol. 4, Issue 5, Sep./Oct. 2012, 3
pages. cited by applicant .
Kim et al., "Nontoxigenic Protein A Vaccine for
Methicillin-Resistant Staphylococcus aureus Infections in Mice," J.
Exp. Med., vol. 207 No. 9, Aug. 30, 2010, pp. 1863-1870. cited by
applicant .
International Search Report and Written Opinion from
PCT/CN2013/088880 dated Sep. 26, 2014. cited by applicant .
Kim et al. Nontoxigenic protein A vaccine for methicillin-resistant
Staphylococcus aureus infections in mice, Journal of Experimental
Medicine, Aug. 30, 2010, vol. 207, No. 9, pp. 1863-1870. cited by
applicant .
Gu et al., "Therapeutic Antibody Drugs on the Control of
Methicillin-resistant Staphylococcus aureus," China Biotechnology,
vol. 32, No. 2, Feb. 15, 2012, pp. 96-99. English Abstract
attached. cited by applicant.
|
Primary Examiner: Hines; Jana A
Attorney, Agent or Firm: Calfee, Halter & Griswold
LLP
Claims
The invention claimed is:
1. A SpA5 protein, wherein the amino acid sequence of the protein
is selected from any one of SEQ ID NO. 1-4.
2. An antibody generated by immunization using the SpA5 protein
according to claim 1.
3. A composition comprising the SpA5 protein according to claim
1.
4. The composition according to claim 3, further comprising one or
more of MntC, HI and mSEB protein.
5. The composition according to claim 4, wherein the amino acid
sequence of the SpA5 protein is selected from any one of SEQ ID NO.
1-4; the amino acid sequence of MntC protein is shown in SEQ ID NO.
13; the amino acid sequence of mSEB protein is shown in SEQ ID NO.
14; and the amino acid sequence of HI protein is shown in SEQ ID
NO. 15 the concentration of each protein is in the range from 10 to
100 .mu.g/ml.
6. A method for preparation of the composition according to claim
3, comprising separately diluting an aluminum adjuvant and a SpA5
protein and mixing the diluted aluminum adjuvant with the diluted
SpA5 protein.
7. The method according to claim 6, which comprises diluting the
individual protein components by the formulation solution
separately, mixing each solution with the aluminium adjuvant
solution diluted by the formulation solution of the same volume,
adsorbing separately, and mixing all the protein solutions
together; or diluting the individual protein components by the
vaccine diluent separately, mixing each solution with the diluted
aluminium adjuvant solution of the same volume separately, mixing
all the protein solutions, and adsorbing together; or mixing all
protein components together, diluting them by the vaccine diluent
and mixing well, mixing the solution with the diluted aluminium
adjuvant solution of the same volume, and adsorbing.
8. The method according to claim 6, wherein the mixing mode for
adsorption is vertical suspension or horizontal suspension at 14
rpm for adsorption of 1 h, at a temperature from 4-37.degree. C.,
the weight ratio between the protein and the element aluminium is
1:1.98; and the aluminium adjuvant is aluminum phosphate or
aluminium hydroxide.
9. The method according to claim 7, wherein the formulation
solution is histidine buffer, comprising 10 mmol/L histidine, 0.02%
poloxamer 188 and 0.9% sodium chloride, pH 6.0.
10. A method for immunizing against Staphylococcus aureus (SA)
infections, comprising immunizing a subject in need thereof with an
effective amount of the SpA5 protein according to claim 1.
11. The method according to claim 10, comprising immunizing the
subject in need at the following time points: Day 0, Day 14, and
Day 21; Day 0, Day 3, and Day 7; or Day 0, Day 3, Day 7, and Day
14.
12. A method for treatment of SA infections, comprising immunizing
a subject in need thereof with an effective amount of the
composition of claim 3.
13. The method according to claim 12, comprising immunizing the
subject in need at the following time points: Day 0, Day 14, and
Day 21; Day 0, Day 3, and Day 7; or Day 0, Day 3, Day 7, and Day
14.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a National Phase Application of International
Application PCT/CN2013/088880, with an international filing date of
Dec. 9, 2013, the entire contents of which is incorporated herein
by reference.
FIELD OF THE INVENTION
The present invention relates to the field of biotechnology, and
particularly to Staphylococcal protein A (SpA) expressed by
Staphylococcus aureus (SA), and more particularly, to a mutant
protein of SpA and the preparation method thereof, and its use in
vaccine formulation.
BACKGROUND OF THE INVENTION
Staphylococcus aureus (SA) is also called "flesh-eating bacteria".
As a representative of gram-positive bacteria, SA is an important
pathogen causing infections in hospitals and communities. SA
infection is characterized by acuteness and purulency. Local
infection may lead to purulent infections of skins and soft tissues
etc, which are unhealed for a long period of time. Systemic
infection may result in severe infections such as osteomyelitis,
septic arthritis, endocarditis, pneumonia, septicopyemia and the
like, and complications. The mortality rate of severe infections
and the complications thereof is as high as 20%. Furthermore,
exotoxins of Staphylococcus aureus will also give rise to fatal
systemic infections, such as food poisoning, scalded skin syndrome
and toxic shock syndrome etc.
With wide and long-term use of antibiotics, bacterial drug
resistance is increasingly severe. As a typical representative,
methicillin-resistant Staphylococcus aureus (MRSA) has become one
of the pathogens with the highest hospital infection rate, such as
ICU infection, post-operation infection, infection of burn wounds
or infection of war wounds etc, since it was first found in 1961.
Additionally, MRSA has also become difficulties in clinical
treatment, due to its strong pathogenicity, wide transmission, easy
outbreak and epidemicity, and multiple-drug resistance, MRSA is
also called "the first super bacteria".
As reported by CDC of the United States, the average annual
population of severe MRSA infections is about 90,000 in America, in
which about 20,000 patients die. As shown by Report of the National
Bacterial Resistance Surveillance of China in 2011: the average
clinical detection rate of MRSA is 60%, in which the extensive
drug-resistance rate is more than 40%. Currently, MRSA, together
with hepatitis B and AIDS, become three major refractory infectious
diseases all over the world, and MRSA takes the first place on the
list. At present, vancomycin is the last effective medicine for
MRSA infections. However, vancomycin-resistant MRSA has already
appeared since 1997, and has overspread globally, leading to a
challenge of "no effective medicine" for MRSA infections.
Due to the severe challenge of "no effective medicine" for
drug-resistant SA infections, "a package plan of 6 policies" has
been proposed by WHO in 2011 to address "drug-resistant bacteria",
and priority will be given to research and development of novel
products for immunological prevention and treatment, such as
inventive vaccines in the future. Accordingly, for effectively
controlling the spread of drug resistance of SA and wide SA
infections in clinical practice, it is strategically and
practically important to study the immunological prevention and
treatment of SA infections, and to develop safe, effective and
novel gene engineering vaccines for SA.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an SpA5 mutant of
SA, and a composition comprising the mutant, as a novel candidate
for SA vaccine.
In the first aspect of the invention, there is provided an SpA5
protein, wherein the amino acid sequence of the protein is selected
from SEQ ID NO. 1-4.
In the second aspect of the invention, there is provided a
nucleotide sequence encoding the SpA5 protein of the invention,
wherein the nucleotide sequence is selected from SEQ ID NO. 9-12,
and a vector and a host comprising the nucleotide sequence.
In the third aspect of the invention, there is provided a method
for preparing the SpA5 protein of the invention.
In the fourth aspect of the invention, there is provided a method
for fermentation of the host comprising SpA5 to prepare the SpA5
protein.
In the fifth aspect of the invention, there is provided a method
for purification of the SpA5 protein of the invention.
In the sixth aspect of the invention, there is provided the use of
the SpA5 protein of the invention as an antigen, and the use of the
SpA5 protein in preparation of a formulation for detection,
prevention or treatment of SA infections.
In the seventh aspect of the invention, there is provided a
polyclonal antibody generated by immunization using the SpA5
protein of the invention, and the use of the polyclonal antibody in
preparation of a formulation for detection, prevention or treatment
of SA infections.
In the eighth aspect of the invention, there is provided a
composition or a kit comprising the SpA5 protein of the invention,
and preferably, the composition is a vaccine.
In the ninth aspect of the invention, there is provided a method
for preparing the composition comprising the SpA5 protein of the
invention.
In the tenth aspect of the invention, there is provided the use of
the composition comprising the SpA5 protein of the invention in
preparation of a formulation for detection, prevention or treatment
of SA infections.
In the eleventh aspect of the invention, there is provided a method
for detecting the contents of individual antigens in the
composition comprising the SpA5 protein of the invention.
In the twelfth aspect of the invention, there is provided a method
for detection, prevention or treatment of SA infections, including
the use of the SpA5 protein of the invention or the composition
comprising the SpA5 protein.
Based on the experiment results, protective immune response can be
effectively triggered in a body by using the SpA5 protein of the
invention as an antigen or using the composition comprising the
SpA5 protein, so as to protect against Staphylococcus aureus
infection with the advantages such as strong immunogenicity, safety
and nontoxicity, high efficacy and quality controllable.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is the map of pGEX vector.
FIG. 2 shows 10% SDS-PAGE analysis of the target proteins
inductively expressed in Example 2; Lane 1: SpA (55 KD); M: middle
range protein molecular weight markers (170, 130, 100, 70, 55, 40,
35, 25, and 15) KD; Lane 2: SpA5wt (33 KD); Lane 3: SpA5ref (KKAA,
33 KD); Lane 4: SpA5 (KKAA, 33 KD); Lane 5: SpA5 (KKVV, 33 KD);
Lane 6: (RRAA, 33 KD); and Lane 7: SpA5 (RRVV, 33 KD).
FIG. 3 illustrates the growth curves of the SpA5 engineering
bacteria in 4 types of culture media.
FIG. 4 shows the expression profiles of SpA5 in M9 and TB culture
media; Lane 1: protein molecular weight markers; Lane 2: expression
profile in M9 culture media; and Lane 3: expression profile in TB
culture media.
FIG. 5 shows the effect of various IPTG concentrations on the
expression of SpA5 protein; Lane 1: protein molecular weight
markers; Lane 2: 0.1 mM; Lane 3: 0.2 mM; Lane 4: 0.5 mM; and Lane
5: 1 mM.
FIG. 6 shows the growth curves of the SpA5 engineering bacteria
with different inoculation amounts before induction.
FIG. 7 shows the effect of different inoculation amounts of the
SpA5 gene engineering bacteria on the expression profiles; Lane 1:
protein molecular weight markers; Lane 2: 5%; Lane 3: 10%; and Lane
4: 15%.
FIG. 8 shows the growth curves of the SpA5 engineering bacteria at
various oxygen concentrations.
FIG. 9 shows the expression profiles of SpA5 protein at various
pO.sub.2 concentrations; Lane 1: protein molecular weight markers;
Lane 2: 25%; Lane 3: 45%; and Lane 4: 65%.
FIG. 10 shows the effect of various glycerol concentrations on the
expression of SpA5 protein; Lane 1: protein molecular weight
markers; Lane 2: 5 mL/L; Lane 3: 10 mL/L; and Lane 4: 15 mL/L.
FIG. 11 shows the expression profile of the target protein at
different inducing temperatures and for different durations; Lane
1: protein molecular weight markers; Lane 2: duration of 0 h; Lane
3: duration of 2 h; Lane 4: duration of 4 h; Lane 5: duration of 6
h; Lane 6: duration of 8 h; and Lane 7: duration of 10 h.
FIG. 12 shows the growth curves of the SpA5 engineering bacteria at
a fermentation volume of 25 L.
FIG. 13 shows the expression profile of the SpA5 protein by the
SpA5 engineering bacteria fermented at the volume of 25 L; Lane 1:
protein molecular weight markers; Lane 2: duration of 0 h; Lane 3:
duration of 1 h; Lane 4: duration of 2 h; Lane 5: duration of 3 h;
Lane 6: duration of 4 h; and Lane 7: duration of 5 h.
FIG. 14 shows the purification of the target protein by
GST-sepharose 4B; Lane M: protein molecular weight markers; Lane 1:
GST-SpA5 fusion protein bound with media; Lane 2: flow-through
fraction; Lane 3: the media bound by the GST-tag, Prescission
Protease (PP) and residue SpA5 after enzyme digestion and elution;
and Lane 4: the target protein SpA5 after enzyme digestion and
elution.
FIG. 15 is the chromatogram illustrating the purification of SpA5
protein on a SP HP column, with two eluting peaks appeared: Peak 1
and Peak 2.
FIG. 16 is the electrophorogram illustrating the purification of
SpA5 protein on a SP HP column; Lane M: protein molecular weight
markers; Lane 1: sample; Lane 2: flow-through fraction; Lane 3 Peak
1; and Lane 4: Peak 2 (eluted by 1 M NaCl).
FIG. 17 is the chromatogram illustrating the purification of SpA5
protein on a MMC column.
FIG. 18 is an electrophorogram illustrating the purification of
SpA5 protein on a MMC column; Lane M: protein molecular weight
markers; Lane 1: loading sample; Lane 2: flow-through fraction;
Lane 3: fraction eluted by 30% solution B; Lane 4: fraction eluted
by 100% solution B.
FIG. 19 is a chromatogram illustrating purification of SpA5 protein
on a Phenyl HP column.
FIG. 20 is an electrophorogram illustrating purification of SpA5
protein on a Phenyl HP column; Lane M: protein molecular weight
markers; Lane 1: elution tube A1; Lane 2: elution tube A2; Lane 3
elution tube A3; Lane 4: elution tube A4; and Lane 5: flow-through
fraction.
FIG. 21 is a chromatogram illustrating purification of SpA5 protein
on a SP HP column (sample conductivity of 4.755 ms/cm).
FIG. 22 is an electrophorogram illustrating purification of SpA5
protein on a SP HP column (sample conductivity of 4.755 ms/cm);
Lane M: protein molecular weight markers; Lane 1: sample; Lane 2:
flow-through fraction; Lane 3: elution tube 1; Lane 4: elution tube
2; Lane 5: elution tube 3; and Lane 6: elution tube 4.
FIG. 23 is a chromatogram illustrating purification of SpA5 protein
on a SP HP column (sample conductivity of 11.622 ms/cm).
FIG. 24 is an electrophorogram illustrating purification of SpA5
protein on an SP HP column (sample conductivity of 11.622 ms/cm);
Lane M: protein molecular weight markers; Lane 1: flow-through
fraction; and Lane 2: F2.
FIG. 25 shows the effect of ammonium sulfate concentration on SpA5
precipitation; Lane 1: the precipitate obtained with 2 M; Lane 2:
the supernatant obtained with 2 M; Lane 3: the supernatant obtained
with 1.6 M; and Lane 4: the precipitate obtained with 1.6 M.
FIG. 26 shows fine purification of SpA mutants; Lane M: protein
molecular weight markers; Lane 1: SpA; Lane 2: SpA5ref (KKAA); Lane
3: SpA5 (KKAA); Lane 4: SpA5 (KKVV); Lane 5: SpA5 (RRAA); and Lane
6: SpA5 (RRVV).
FIG. 27 is a chromatogram illustrating purification of SpA5 protein
on a SP HP column (batch I) at a scaled-up level.
FIG. 28 is a chromatogram illustrating purification of SpA5 protein
on a Q HP column (batch I) at a scaled-up level.
FIG. 29 is an electrophorogram illustrating purification of SpA5
protein at a scaled-up level (batch I); Lane M: protein molecular
weight markers; Lane 1: sample before purification; Lane 2:
flow-through fraction; Lane 3: eluent from a SP HP column; Lane 4:
sample of re-dissolved precipitate obtained with
(NH.sub.4).sub.2SO.sub.4; Lane 5: eluent from a G25 column; and
Lane 6: flow-through fraction from a Q HP column.
FIG. 30 is a chromatogram illustrating purification of SpA5 protein
on a SP HP column (batch II) at a scaled-up level.
FIG. 31 is a chromatogram illustrating purification of SpA5 protein
on a Q HP column (batch II) at a scaled-up level.
FIG. 32 is an electrophorogram illustrating purification of SpA5
protein at a scaled-up level (batch II); Lane M: protein molecular
weight markers; Lane 1: sample before purification; Lane 2: eluent
from a SP HP column; Lane 3: sample of re-dissolved precipitate
obtained with (NH.sub.4).sub.2SO.sub.4; Lane 4: eluent from a G25
column; and Lane 6: flow-through fraction from a Q HP column.
FIG. 33 is a chromatogram illustrating purification of SpA5 protein
on a SP HP column (batch III) at a scaled-up level.
FIG. 34 is a chromatogram illustrating purification of SpA5 protein
on a Q HP column (batch III) at a scaled-up level.
FIG. 35 is an electrophorogram illustrating purification of SpA5
protein at a scaled-up level (batch III); Lane M: protein molecular
weight markers; Lane 1: sample before purification; Lane 2:
flow-through fraction; Lane 3; eluent from a SP HP column; Lane 4:
eluent from a G25 column; and Lane 5: flow-through fraction from a
Q HP column.
FIG. 36 shows the detection of SpA5 (KKAA) mutant by HPLC; the
retention time of the main peak: 13.282 min; and the main peak area
ratio: 98.2%.
FIG. 37 shows the binding capacities of SpA and each SpA5 protein
to human IgG by ELISA (mean value.+-.stdev, n=12).
FIG. 38 shows the F(ab).sub.2 fragments after enzyme digestion and
purification; Lane 1: F(ab).sub.2; Lane M: protein molecular weight
markers; and Lane 2: the antibody.
FIG. 39 shows the reduction of binding capacities of SpA to human
IgG caused by antibody F(ab).sub.2 fragments produced in the
rabbits immunized by each SpA5 protein tested using ELISA (mean
value.+-.stdev, n=5).
FIG. 40 shows the induced apoptosis of spleen B lymphocytes of the
mice after immunization by SpA and each SpA5 protein.
FIG. 41 shows 12% SDS-PAGE analysis before and after SpA5
adsorption by aluminum phosphate; Lane 1: the supernatant obtained
by centrifugation after adsorption by aluminum phosphate: SpA5ref
(KKAA); Lane 2: the supernatant obtained by centrifugation after
adsorption by aluminum phosphate: SpA5 (KKAA); Lane 3: the
supernatant obtained by centrifugation after adsorption by aluminum
phosphate: SpA5 (RRVV); and Lane 4: protein solution (using SpA5
(KKAA) as a control).
FIG. 42 is a graph showing the geometric mean antibody titer in the
serum of the animal in each group.
FIG. 43 shows SDS-PAGE analysis of the samples of Example 19; Lane
M: protein molecular weight markers; Lane 1: protein solution (free
of aluminum phosphate adjuvant) formulated of the same
concentration in Example 19; Lane 2: the sample obtained from the
semi-finished product in Example 16 after centrifugation and
dissociation; Lane 3: the sample obtained from the supernatant of
the semi-finished product in Example 16 after centrifugation; Lane
4: the sample obtained from the semi-finished product in Example 17
after centrifugation and dissociation; Lane 5: the sample obtained
from the supernatant of the semi-finished product in Example 17
after centrifugation; Lane 6: the sample obtained from the
semi-finished product in Example 18 after centrifugation and
dissociation; and Lane 7: the sample obtained from the supernatant
of the semi-finished product in Example 18 after
centrifugation.
FIG. 44 shows SDS-PAGE analysis of the samples of Example 21; Lane
M: protein molecular weight markers; Lane 1: 80 .mu.g HI protein
sample; Lane 2: 120 .mu.g HI protein sample; Lane 3: 160 .mu.g HI
protein sample; Lane 4: SpA5 (KKAA) free of adjuvant; Lane 5: 80
.mu.g SpA5 (KKAA); Lane 6: 120 .mu.g SpA5 (KKAA); and Lane 7: 160
.mu.g SpA5 (KKAA).
FIG. 45 shows SDS-PAGE analysis of the samples of Example 21; Lane
M: protein molecular weight markers; Lane 1: mSEB protein free of
adjuvant; Lane 2: 80 .mu.g mSEB protein; Lane 3: 80 .mu.g mSEB
protein; Lane 4: 160 .mu.g mSEB protein; Lane 5: MntC protein free
of adjuvant; Lane 6: 80 .mu.g MntC protein; Lane 7: 120 .mu.g MntC
protein; and Lane 8: 160 .mu.g MntC protein.
FIG. 46 shows SDS-PAGE analysis of the samples of Example 22; Lane
M: protein molecular weight markers; Lane 1: randomly collected
sample 1--the supernatant after centrifugation; Lane 2: randomly
collected sample 1--dissociated precipitate after centrifugation;
Lane 3: randomly collected sample 2--the supernatant after
centrifugation; Lane 4: randomly collected sample 2--dissociated
precipitate after centrifugation; Lane 5: randomly collected sample
3--the supernatant after centrifugation; and Lane 6: randomly
collected sample 3--dissociated precipitate after
centrifugation.
FIG. 47 shows SDS-PAGE analysis of the samples of Example 22; Lane
M: protein molecular weight markers; Lane 1: randomly collected
sample 4--the supernatant after centrifugation; Lane 2: randomly
collected sample 4--dissociated precipitate after centrifugation;
Lane 3: randomly collected sample 5--the supernatant after
centrifugation; Lane 4: randomly collected sample 5--dissociated
precipitate after centrifugation; Lane 5: randomly collected sample
6--the supernatant after centrifugation; and Lane 6: randomly
collected sample 6--dissociated precipitate after
centrifugation.
FIG. 48 shows SDS-PAGE analysis of the samples of Example 24; Lane
1: the supernatant obtained by centrifugation after 0.5 h
adsorption of HI protein; Lane 2: the supernatant obtained by
centrifugation after 1 h adsorption of HI protein; Lane 3: the
supernatant obtained by centrifugation after 2 h adsorption of HI
protein; Lane 4: the supernatant obtained by centrifugation after 4
h adsorption of HI protein; Lane 5: the supernatant obtained by
centrifugation after 8 h adsorption of HI protein; Lane 6: the
supernatant obtained by centrifugation after 0.5 h adsorption of
SpA5 (KKAA); Lane 7: the supernatant obtained by centrifugation
after 1 h adsorption of SpA5 (KKAA); Lane 8: the supernatant
obtained by centrifugation after 2 h adsorption of SpA5 (KKAA);
Lane 9: the supernatant obtained by centrifugation after 4 h
adsorption of SpA5 (KKAA); and Lane 10: the supernatant obtained by
centrifugation after 8 h adsorption of SpA5 (KKAA).
FIG. 49 shows SDS-PAGE analysis of the samples of Example 24; Lane
1: the supernatant obtained by centrifugation after 1 h adsorption
of MntC protein; Lane 2: the supernatant obtained by centrifugation
after 2 h adsorption of MntC protein; Lane 3: the supernatant
obtained by centrifugation after 4 h adsorption of MntC protein;
Lane 4: the supernatant obtained by centrifugation after 8 h
adsorption of MntC protein; Lane 5: the supernatant obtained by
centrifugation after 1 h adsorption of mSEB protein; Lane 6: the
supernatant obtained by centrifugation after 2 h adsorption of mSEB
protein; Lane 7: the supernatant obtained by centrifugation after 4
h adsorption of mSEB protein; and Lane 8: the supernatant obtained
by centrifugation after 8 h adsorption of mSEB protein.
FIG. 50 shows SDS-PAGE analysis of the samples in Example 25 stored
for 4 weeks; Lane M: protein molecular weight markers; Lane 1:
randomly collected sample 1--the supernatant after centrifugation;
Lane 2: randomly collected sample 1--dissociated precipitate after
centrifugation; Lane 3: randomly collected sample 2--the
supernatant after centrifugation; Lane 4: randomly collected sample
2--dissociated precipitate after centrifugation; Lane 5: randomly
collected sample 3--the supernatant after centrifugation; and Lane
6: randomly collected sample 3--dissociated precipitate after
centrifugation.
FIG. 51 shows SDS-PAGE analysis of the samples in Example 25 stored
for 8 weeks; Lane M: protein molecular weight markers; Lane 1:
randomly collected sample 1--the supernatant after centrifugation;
Lane 2: randomly collected sample 1--dissociated precipitate after
centrifugation; Lane 3: randomly collected sample 2--the
supernatant after centrifugation; Lane 4: randomly collected sample
2--dissociated precipitate after centrifugation; Lane 5: randomly
collected sample 3--the supernatant after centrifugation; and Lane
6: randomly collected sample 3--dissociated precipitate after
centrifugation.
FIG. 52 shows SDS-PAGE analysis of the samples in Example 25 stored
for 12 weeks; Lane M: protein molecular weight markers; Lane 1:
randomly collected sample 1--the supernatant after centrifugation;
Lane 2: randomly collected sample 1--dissociated precipitate after
centrifugation; Lane 3: randomly collected sample 2--the
supernatant after centrifugation; Lane 4: randomly collected sample
2--dissociated precipitate after centrifugation; Lane 5: randomly
collected sample 3--the supernatant after centrifugation; and Lane
6: randomly collected sample 3--dissociated precipitate after
centrifugation.
FIG. 53 illustrates the change of body weight of the male animals
in each group at various time points.
FIG. 54 illustrates the change of body weight of the female animals
in each group at various time points.
FIG. 55 is a plot showing preliminary investigation on the survival
rate vs the infective dose of Staphylococcus aureus.
FIG. 56 is a plot that determines the infective dose in a pneumonia
model.
FIG. 57 illustrates the counts in both lungs in C57BL/6J mice after
infection by SA77.
FIG. 58 illustrates pulmonary tissue sections in experiment group
(100.times., A for control group, and B for experiment group).
FIG. 59 is a graph that determines the dissociation time between
the antigen and the adjuvant in Example 38; Lane M: protein
molecular weight markers, and Lane 1, 2 and 3 showing the results
at the dissociation time of 10, 20 and 30 min, respectively.
FIG. 60 is a graph showing the comparison between dissociation
using sodium carbonate and other methods: Lane M: protein molecular
weight markers, and Lane 1, 2, 3 and 4 representing dissociation
using sodium citrate, guanidinium hydrochloride, citric acid and
sodium carbonate, respectively.
FIG. 61 shows the antigenicity of the vaccines detected by
western-blot in Example 40.
FIG. 62 is standard curves plotted between the concentrations of 4
standard antigens vs grey values in Example 40.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in detail with respect to
the following particular embodiments, but the invention is not
limited thereto.
A. SpA5 Mutant
SpA can be expressed by most clinical SA isolates. SA has a
molecular weight of 55 kDa, and locates on the cell wall. The
precursor of SpA contains an N-terminal signal peptide and a
C-terminal screening signal, allowing SpA covalently bound to the
cell wall. The N-terminal segment of the mature peptide contains 5
Ig binding domains consisting of 56-61 amino acid residues, which
further wind into 3 .alpha.-helix bundles. These domains can bind
mammalian IgG, and destroy antibody opsonophagocytosis; the domains
also can bind VH3-type B cell receptors, leading to the death of
B-cells, followed by breaking down acquired and innate immune
response. Accordingly, it is an important choice for the anti-MRSA
vaccines to induce anti-SpA antibodies in a body, block immunologic
escape mechanism of MRSA. However, the intended objective can not
be implemented by using natural SpA as an antigen, due to its
binding capacity to antibodies. Accordingly, mutant SpA is
demanded, which removes its antibody binding capacity and retains
its immunogenicity.
The precursor of SpA protein of ATCC international standard strain
MRSA 252 of Staphylococcus aureus (named as SpA (252), SEQ ID NO:5)
includes 516 amino acids, in which the first 36 amino acids
constitute a signal peptide sequence, and the amino acids at
position 37-327 form 5 Ig binding domains including EDABC, having
totally 291 amino acids (named as SpA (252). Ig binding domain is
an active region of SpA, and also the research highlight. In order
to obtain highly immunoreactive vaccines, mutation has been
performed to Ig binding domains of the SpA from S. aureus Newman
strain, and a certain effect has been brought about, as described
in Kim H K et al. (Nontoxigenic protein A vaccine for
methicillin-resistant Staphylococcus aureus infections in mice. J
Exp Med. 2010 Aug. 30; 207(9):1863-70).
Based on the prior art, Ig binding domains of the SpA protein in
ATCC international standard strain of Staphylococcus aureus MRSA252
have been cloned and mutated in the present invention. After
activity assay, it has been found that the SpA5 protein of the
invention has higher activity than that of the SpA mutant protein
provided in the reference.
In the present invention, 43Q, 44Q, 96Q, 97Q, 162Q, 163Q, 220Q,
221Q, 278Q, and 279Q of SpA (252) have been replaced by K or R at
the same time, and 70D, 71D, 131D, 132D, 189D, 190D, 247D, 248D,
305D, and 306D have been replaced by A or V at the same time,
followed by modification, 4 SpA5 proteins, including SpA5 (KKAA)
(SEQ ID NO. 1), SpA5 (RRAA) (SEQ ID NO. 2), SpA5 (KKVV) (SEQ ID NO.
3), and SpA5 (RRVV) (SEQ ID NO. 4), have been obtained in the
present invention. In these 4 proteins, amino acids at position
6-296 belong to SpA (252), and the first 5 amino acids (GPLGS, SEQ
ID NO. 8) come from the modified amino acids of the expression
vector, the encoding sequence of which is preferably
gggcccctgggatcc (SEQ ID NO. 16).
Accordingly, in the present invention, there are provided
nucleotide sequences encoding any one of the SpA5 proteins of SEQ
ID NO. 1-4. Based on known amino acid sequences, appropriate
nucleotide sequences encoding the amino acid sequences can be
designed by the one skilled in the art as desired, and the designed
nucleotide sequences also can be expressed.
In a preferable embodiment, the nucleotides sequences are shown in
SEQ ID NO. 9-12.
In another aspect, the present invention provides a vector or a
host comprising the nucleotide sequences encoding the SpA5 protein
of the invention.
The vector can be any vectors that are suitable for protein
expression. In preferable embodiments of the invention, the vector
is expression vector pGEX or pET series vector, and more
preferably, is expression vector pGEX-6P-2. The map of pGEX series
vector is shown in FIG. 1.
pGEX-6p-2 vector preferably employed in the present invention has
an advantage that a 26 kDa glutathione-S-transferase (GST) is
attached, so that the expressed fusion protein contains a GST-tag
which facilitates protein purification. As compared with other
fusion vectors, pGEX series vector has the following advantages:
mild purification conditions, simple process, and no denaturant
added, which allow the proteins to retain their conformation and
immunogenicity as much as possible after purification.
The host can be any expression cells known to the one skilled in
the art, and preferably, is E. coli XL1-Blue.
In another aspect, there is provided a method for preparing the
SpA5 protein, in which the method can be chemical synthesis or gene
expression. The method for preparing the SpA5 protein of the
invention can be completely understood by the one skilled in the
art based on the sequence of the SpA5 protein and the common
knowledge in the field.
In another aspect, the present invention provides a fermentation
process for the SpA5 gene engineering bacteria, which comprises
inoculating a certain amount of seed bacteria into the fermentation
media containing glycerol and a certain amount of dissolved oxygen,
and inducing for a certain period of time by an inductive agent to
express the recombinant protein. Preferably, several factors are
involved in the process, including the culture media, the
inoculation amount of seed bacteria, the amount of glycerol and
dissolved oxygen in the media, the type and the concentration of
inductive agent, and the temperature and the duration for
induction. More preferably, the culture media is animal origin TB,
animal origin M9, plant origin TB, and plant origin M9, and
preferably, is animal origin TB. The inoculation amount of seed
bacteria is in the range from 5% to 15%, and preferably is 10%. The
amount of glycerol is from 5 to 15 mL/L medium, and preferably, is
10 mL/L medium. The amount of dissolved oxygen is 25%-65%, and
preferably, is 45%. The inductive agent can be lactose or IPTG and
preferably, is IPTG The concentration of the inductive agent is 200
.mu.mol/L-1 mmol/L medium, and preferably, is 200 .mu.mol/L medium.
The temperature for induction is 16-37.degree. C., and preferably,
is 30.degree. C. The duration for induction is 1-6 h, and
preferably, is 5 h.
In a particular embodiment of the fermentation process, the basal
medium is animal origin TB medium, in which the amount of glycerol
is 10 mL/L; at the beginning of fermentation, the seed bacteria is
inoculated in a ratio of 10%; during the course of fermentation,
the amount of dissolved oxygen is maintained at around 45% all the
time; during induction, the temperature is set at 30.degree. C.,
the concentration of IPTG is 0.2 mM, and the duration for induction
is 5 h.
In another aspect, the present invention further provides a method
for purification of the SpA5 protein after host bacteria (gene
engineering bacteria) fermentation, which comprises 5 sequential
steps, including affinity chromatography, cation-exchange
chromatography, ammonium sulfate precipitation, desalinization and
anion-exchange chromatography. In the purification process, the
affinity chromatography includes digestion by Prescission Protease
(PP enzyme) after binding by GST affinity chromatography media, and
preferably, by GST sepharose 4B media; the media for
cation-exchange chromatography is SP HP, Capto MMC, and Phenyl HP,
and preferably is SP HP; the media for desalinization is G 25; and
the media for anion-exchange chromatography is Q HP.
The equilibrium buffer used for cation-exchange chromatography is
10-20 mM PB, pH 6.5-7.5, and preferably, 20 mM PB, pH 7.0. The
elution buffer used for cation-exchange chromatography is 10-20 mM
PB+0.3-1.5 M NaCl, pH 6.5-7.5, and preferably, 20 mM PB+1 M NaCl,
pH 7.0. Preferably, the elution procedure is a linear gradient of
0-50% elution buffer for 5 column volumes.
The procedure for ammonium sulfate precipitation includes mixing
the sample with 3 M (NH.sub.4).sub.2SO.sub.4 in a ratio of
1:2-1.4:1.6 (V/V) at 4.degree. C., followed by stirring for 10 min
and centrifugation at 6000 r/min for 20 min; and preferably, the
ratio between the sample and ammonium sulfate is 1.4:1.6.
The equilibrium buffer used for the anion-exchange chromatography
is 5-15 mM His+0.01-0.05% poloxamer 188+0.9% NaCl, pH 5-7; and
preferably, 10 mM His+0.01% poloxamer 188+0.9% NaCl, pH 6.0.
Preferably, the host (gene engineering bacteria) is disrupted
before affinity chromatography, so that the protein can be
released.
In another aspect, the present invention provides the use of the
SpA5 protein in detection, prevention or treatment of SA
infections, or in preparation of a formulation for detection,
prevention or treatment of SA infections. Preferably, the
formulation is a vaccine.
In another aspect, the present invention further provides an
antibody produced by the SpA5 protein immunization, in which the
antibody is a polyclonal antibody, that can be used for detection,
prevention and treatment of SA infection-associated diseases, or
used for preparation of corresponding formulations.
In another aspect, the present invention further provides a
composition or a kit comprising the SpA5 protein of the invention.
The composition can be a reagent or medicine (such as a vaccine)
for prevention, detection or treatment of SA infections. The kit
can be any types of kits known in the field, such as detection kit
or treatment kit, etc.
In another aspect, the present invention provides a method for
detection, prevention or treatment of SA infections, including the
use of the SpA5 protein of the invention or the composition
comprising the SpA5 protein. Preferably, the subject in need of the
method is immunized at the following time points: Day 0, Day 14,
and Day 21; Day 0, Day 3, and Day 7; Day 0, Day 3, Day 7, and Day
14.
EXAMPLES
All reagents and strains used in Examples were shown as follows.
Any chemical reagent whose manufacturer was not specified in
Examples of the present application could be purchased from
conventional chemical or biological suppliers in the field.
1. Strains
ATCC international standard strain MRSA-252 of Staphylococcus
aureus, supplied by ATCC, US;
Host strain Escherichia coli XL1-blue, a product of Agilent,
US.
2. Plasmid
Plasmid pGEX-6p-2, a product of GE Healthcare, US.
3. Reagents
PrimeSTAR HS DNA-polymerase, DNA molecular weight markers, BamH I
and Not I restriction enzyme, protein molecular weight markers, and
DNA ligase were purchased from TakaRa (Dalian, China);
Plasmid extraction kit and gel recovery kit were purchased from
Omega corporation, US;
Rest reagents, including agar powder and Tween-20, were purchased
from domestic companies;
MH medium, supplied by Beijing Aoboxing Biotechnology Co., Ltd.
(2.0 g powdered beef, 1.5 g soluble starch, and 17.5 g acid
hydrolyzed casein), was added into water to a final volume of 1 L,
pH 7.4.+-.0.2;
MH plate: agar was added to MH medium with a final concentration of
1.5 g/100 mL;
PBS: potassium dihydrogen phosphate (KH.sub.2PO.sub.4) 0.2 g
(Domestic reagent of analytical grade), disodium hydrogen phosphate
(Na.sub.2HPO.sub.4.12H.sub.2O) 2.9 g (Domestic reagent of
analytical grade), sodium chloride (NaCl) 8.0 g (Domestic reagent
of analytical grade), and potassium chloride (KCl) 0.2 g, were
added into water to a final volume of 1000 mL, pH 7.4;
20 mM PB buffer: potassium dihydrogen phosphate (KH.sub.2PO.sub.4)
0.2 g, disodium hydrogen phosphate (Na.sub.2HPO.sub.4.12H.sub.2O)
2.9 g, and potassium chloride (KCl) 0.2 g, were added into water to
a final volume of 1000 mL, pH 7.0;
Ampicillin, and kanamycin (North China Pharmaceutical Co.
Ltd.);
5.times. Loading buffer (250 mM Tris-HCl (pH 6.8), 10% (W/V) SDS,
0.5% (W/V) bromphenol blue, 50% (V/V) glycerol, and 5% (W/V)
.beta.-mercaptoethanol);
Glutathione sepharose 4B (GE Healthcare, US);
Aluminum phosphate adjuvant: GENERAL CHEMICAL corporation (US) (20
mg/ml, concentration of element aluminium: 5.3 mg/ml);
Vaccine diluent: 10 mM histidine (Merck corporation, US,
pharmaceutical grade), 0.9% NaCl (Sichuan Kelun Corporation,
physiological saline for injection) and 0.01% poloxamer 188 (Merck
corporation, US, pharmaceutical grade), pH 6.0, pyrogen-free.
Example 1: Sequence Synthesis
Base sequence and amino acid sequence of SpA (SpA (252), SEQ ID NO.
5) were obtained using MRSA252 genome (GI:49240382) as the templet.
Based on the preference of E. coli, rare codon analysis and
optimization were performed on website:
http://people.mbi.ucla.edu/sumchan/caltor.html for the bases in 5
domains, including E, D, A, B, and C (namely, 37aa-327aa of SEQ ID
NO.5, referred as SpA5 (252). Point mutation was performed for 4
amino acids in each domain, and the nucleotide sequences encoding
the amino acid sequence of position 6-296 of 4 proteins: SpA5
(KKAA), SpA5 (RRAA), SpA5 (KKVV), and SpA5 (RRVV) were synthesized.
The nucleotide sequences were separately linked to expression
vector pGEX-6P-2 using BamH I and Not I as two cleavage sites. Host
XL-1 Blue was then transformed by the vector (performed by Shanghai
Generay Biotech Co., Ltd). The corresponding nucleotide sequences
of four SpA5 proteins were shown in SEQ ID NO. 9-12.
In the same manner, the nucleotide sequence of SpA5 (252) was
synthesized, linked to the expression vector and transformed into
the host.
For the convenience of comparison with the SpA mutant which has
been reported, the nucleotide sequence encoding the amino acid
sequence of SpA5ref (KKAA) (SEQ ID NO. 6) at position 6-301 was
synthesized based on the amino acid sequence of SpA5ref reported
(Nontoxigenic protein A vaccine for methicillin-resistant
Staphylococcus aureus infections in mice. Kim H K, Cheng A G, Kim H
Y, Missiakas D M, Schneewind O. J Exp Med. 2010 Aug. 30;
207(9):1863-70). The nucleotide sequence was then linked to the
expression vector using the above method, and transformed into the
host.
In this example, all synthesized genes were inserted into BamH I
site, and subsequently transformed into the host. After expressed
by the recombinant engineering bacteria and digested by PP enzyme
(GE corporation, US) (5 amino acids (GPLGS) of the vector were
retained at N-terminus after digestion), proteins used for the
following experiments were obtained, including SpA5 (KKAA), SpA5
(RRAA), SpA5 (RRVV), and SpA5 (KKVV) of the present invention, a
protein for comparison SpA5wt (sequence shown in SEQ ID NO. 7,
namely, the product obtained by linking the nucleotide sequence of
SpA5 (252) to vector pGEX-6P-2, transformed into XL-1Blue and
expressed, and finally digested by PP enzyme, which had 5 more
amino acids GPLGS (SEQ ID NO.8) at N-terminus than SpA5 (252) and
SpA5ref (KKAA).
Example 2: Inductive Expression in Escherichia coli, Purification
and Characterization of Each Protein
1) Inductive Expression of the Target Proteins
(1) 100 .mu.L culture solution of each recombinant engineering
bacteria was collected and added into 10 mL LB medium containing
ampicillin at a concentration of 100 .mu.g/mL, incubated at
37.degree. C. at 80 rpm overnight. 400 .mu.L culture solution
incubated overnight was withdrawn and added to 20 mL LB medium
containing ampicillin at a concentration of 100 .mu.g/mL, inbucated
at 37.degree. C. for 2-3 h at 220 rpm. After OD.sub.600mn increased
to 0.8-1.0 in the second activation, IPTG was added to a final
concentration of 200 .mu.M. The culture was subsequently placed in
a shaker at 30.degree. C. for 3 h, and then at 16.degree. C.
overnight for inductive expression.
(2) After inductive expression, the bacteria solution was
centrifugated at 10000 rpm for 5 min. The supernatant after
centrifugation was discarded, and 1 mL PBS was added and mixed
well. Then the bacteria were disrupted by ultrasound (power of 300
W) for 10 min (in a cycle of 6 s on and 9 s off). Subsequently, the
mixture was centrifugated at 14000.times.G for 15 min at 4.degree.
C. to separate the supernatant from the precipitate.
2) Treatment of the Supernatant
Binding of the supernatant: 20 .mu.L glutathione-sepharose 4B (GE
Healthcare, US) was washed with PBS for 3 times, then the prepared
supernatant was added, followed by binding at room temperature for
1 h. The mixture was then centrifugated at 14000 rpm for 3 min at
4.degree. C., then was washed by PBS-0.25% Tween 20 twice and by
PBS once.
According to manufacturer's recommended instructions, the bound
protein was digested using PP enzyme (Prescission protease, GE
Healthcare, US) and eluted. The PP enzyme used had a GST-tag, which
facilitated its removal. After digestion, the supernatant was
collected after centrifugation. 16 .mu.L supernatant was added to 4
.mu.L 5.times. loading buffer, and boiled for 5 min. The solution
was then centrifugated at 14000 rpm for 3 min.
3) 10% SDS-PAGE
The electrophoretogram was shown in FIG. 2. Lane 1 illustrated SpA
(252) (1.5 .mu.g) purchased from Invitrogen Corporation (US) (REC
PROTEIN A10-1100). As shown in FIG. 2, the protein was
purified.
Example 3: Fermentation of the Engineering Bacteria
1. Determination of the Conditions for Fermentation
1) Effect of the Medium on the Growth of Engineering Bacteria and
the Expression of Target Protein
4 types of culture media were tested by shaking culture using the
method as described in Example 2:
Modified TB medium of plant origin (potassium dihydrogen phosphate
2.3 g, disodium hydrogen phosphate dodecahydrate 13 g, glycerol 5
mL, yeast extract 24 g, soybean peptone 12 g, and magnesium sulfate
1 g, adding water to a final volume of 1 L);
Modified TB medium of animal origin (potassium dihydrogen phosphate
2.3 g, disodium hydrogen phosphate dodecahydrate 13 g, glycerol 5
mL, yeast extract 24 g, animal origin tryptone 12 g, and magnesium
sulfate 1 g, adding water to a final volume of 1 L);
Plant origin M9-CAA medium (disodium hydrogen phosphate 15.6 g,
potassium dihydrogen phosphate 4.3 g, ammonium chloride 1 g,
magnesium sulfate 1 g, sodium chloride 0.67 g, glucose 5 g, soybean
peptone 3.6 g, plant origin yeast powder 4 g, and acid hydrolyzed
casein 6 g, adding water to a final volume of 1 L);
Animal origin M9-CAA medium (disodium hydrogen phosphate 15.6 g,
potassium dihydrogen phosphate 4.3 g, ammonium chloride 1 g,
magnesium sulfate 1 g, sodium chloride 0.67 g, glucose 5 g, animal
tryptone 3.6 g, animal origin yeast powder 4 g, and acid hydrolyzed
casein 6 g, adding water to a final volume of 1 L);
SpA5 (KKAA) engineering bacteria were inoculated to an Amp.sup.+
(100 .mu.g/mL) LB agar plate, and incubated at 37.degree. C. for
16-20 h. Individual bacterial colonies were picked up and
inoculated to 10 mL Amp.sup.+LB medium. The suspension was
incubated at 37.degree. C. in a shaker shaken at 200 rpm until
OD.sub.600 increased to about 2. The suspension was then inoculated
to 4 types of culture media (100 mL each) in a ratio of 1:100, then
incubated at 37.degree. C. at 200 rpm for 14 h. Samples were
collected at an interval of 2 h to detect OD.sub.600. The results
were shown in FIG. 3.
As shown in FIG. 3, the growth in the plant origin culture media
was poorer than that in the animal origin culture media. In the
plant origin culture media, the growth started to slow down at
about 8 h, whereas in the animal origin culture media, the growth
remained in exponential phase all the time. In another aspect, the
growth in the TB medium was better than that in the M9 medium.
Thus, the animal origin culture media were selected for the
following experiments (animal origin modified TB medium and M9-CAA
medium were hereafter referred as TB medium and M9 medium,
respectively).
Fresh bacteria solution of the SpA5 engineering bacteria
(OD.sub.600 of about 2) was inoculated to 100 mL TB or M9 medium in
a ratio of 1:100 respectively. The culture was then incubated at
37.degree. C. at 200 rpm until OD.sub.600 increased to about 0.8.
Subsequently, 1 mM IPTG was added, and the expression was induced
at 25.degree. C. for 12 h. 100 mL bacteria solution was then
centrifugated, the supernatant was discarded and the precipitate
was weighed: 2.4 g for TB medium and 1.5 g for M9 medium. To the
precipitate, PBS was added in a ratio of 1 g:10 mL, then the
bacteria were disrupted by ultrasound (power of 300 W) for 10 min
(in a cycle of 6 s on and 9 s off). Subsequently, the protein was
bound to GST 4B (as detailedly described in Example 4). The result
was analyzed by 10% SDS-PAGE, and shown in FIG. 4.
As shown in FIG. 4, the TB medium was better than the M9 medium in
terms of the amount of the expressed target protein. Besides, the
TB medium was also more favorable due to the wet weight of
bacteria. Thus, the TB medium was selected as the basal medium for
the fermentation of the SpA5 engineering bacteria.
2) Effect of IPTG Concentration on the Expression of Target
Protein
The effect of different IPTG concentrations on the expression level
of target protein was studied. The optimal IPTG concentration was
selected after comparison among various final concentrations,
including 0.1 mM, 0.2 mM, 0.5 mM, and 1 mM. Fresh bacteria solution
of the SpA5 engineering bacteria (OD.sub.600 of about 2) was
inoculated to 4 flasks of TB medium (100 mL each) in a ratio of
1:100, then incubated at 37.degree. C. at 200 rpm until OD.sub.600
increased to about 0.8. Subsequently, IPTG was added at four
different concentrations (1 concentration for 1 flask), and the
expression was induced at 25.degree. C. for 12 h. The bacteria
solution in each flask was centrifugated separately, and the
supernatant was discarded. To the precipitate, PBS was added in a
ratio of 1 g:10 mL, then disrupted by ultrasound (power of 300 W)
for 10 min (in a cycle of 6 s on and 9 s off). Subsequently, the
protein was bound to GST 4B. The result was analyzed by 10% SD
S-PAGE, and shown in FIG. 5.
As shown in FIG. 5, the expression induced by IPTG at the
concentration of 0.2 mM was obviously better than that at 0.1 mM,
and was substantially the same as that at 0.5 mM or 1 mM. Thus, 0.2
mM was selected as the final concentration of IPTG for
fermentation.
3) Effect of the Inoculation Amount on the Growth of the
Engineering Bacteria and the Expression of Target Protein
The effect of 3 different inoculation amounts, including 5%, 10%,
and 15%, on the fermentation process was studied. The optimal
inoculation amount was determined according to the growth curve and
the amount of the expressed protein. The seed bacteria (OD.sub.600
of about 2) was poured into a fermentation tank and begin timing.
Samples were collected every one hour for OD.sub.600 detection
until induction is started. The growth curve at the initial stage
was plotted for the engineering bacteria (FIG. 6). Based on the
plotted curve, the growth rate can be determined. After induction,
samples were treated in the same manner as previously described.
10% SDS-PAGE was performed to determine the amount of expressed
protein (FIG. 7).
As shown in FIG. 6, the growth rate for the inoculation amount of
5% was the slowest, and its highest OD.sub.600 was much lower than
that obtained for the other 2 inoculation amounts. While for the
inoculation amounts of 10% and 15%, no significant difference was
observed.
As shown in FIG. 7, the amount of the expressed target protein
varied among different inoculation amounts. The largest expression
amount was achieved at the inoculation amount of 5%, follow by 10%,
with slight difference. The smallest expression amount was seen for
the inoculation amount of 15%.
In conclusion, the expression amount for the inoculation amount of
5% was the best, however, the growth rate was slow and the yield of
the bacteria was little under this condition. For the inoculation
amount of 15%, the expression amount was the smallest, although its
growth rate was fast. While for the inoculation amount of 10%, the
expression amount was almost the same as that obtained for the
inoculation amount of 5%, and the growth rate was also as fast as
that for the inoculation amount of 15%. Thus, the inoculation
amount of 10% was selected for the fermentation.
4) Effect of the Oxygen Concentration on the Growth of the
Engineering Bacteria and the Expression of Target Protein
The engineering bacteria were a kind of facultative aerobe, so the
oxygen concentration was crucial to its growth. Thus, it was
important to control the amount of dissolved oxygen during the
course of fermentation. In this experiment, the growth of the
bacteria (FIG. 8) and the amount of the expressed target protein
(FIG. 9) were studied when the dissolved oxygen amount was set at
25%, 45%, or 65%.
It was suggested by the result that the growth of the bacteria was
the best at the dissolved oxygen amount of 45%; while for the
amount of the expressed protein, the dissolved oxygen amount of 45%
was also the best. Thus, the dissolved oxygen amount of 45% was
selected for the fermentation process.
5) Determination of the Amount of Glycerol Used
The expression amount of SpA5 varied when induced at different
amounts of the bacteria, which were further affected directly by
the amount of glycerol in the medium. When the glycerol in the tank
was completely consumed, the growth of bacteria will stop, the pH
value and the dissolved oxygen amount would increase rapidly in a
short period of time, at which time the induction should be started
immediately by adding IPTG The amount of glycerol decided the
timing of induction directly. The best timing for induction was
determined based on the effect of glycerol dosage, including 5
mL/L, 10 mL/L, and 15 mL/L, on the expression of the target protein
and the yield of the wet weight of bacteria.
FIG. 10 showed the amounts of the expressed target protein at 3
different glycerol dosages. As shown in FIG. 10, the largest
expression amount was observed at the glycerol dosage of 5 mL/L,
followed by the dosage of 10 mL/L, with slight difference; while
the smallest expression amount was observed at the glycerol dosage
of 15 mL/L. However, at the glycerol dosage of 5 mL/L, the wet
weight of the bacteria was much lower; while for both glycerol
dosages of 10 mL/L and 15 mL/L, the wet weights were almost the
same. Thus, the glycerol dosage was determined at 10 mL/L.
6) Effect of the Induction Temperature and Duration on the
Expression of the Target Protein
The expression of the target protein was studied at 3 different
induction temperatures, including 30.degree. C., 25.degree. C., and
16.degree. C. Besides, the duration used to achieve the maximal
expression amount was also studied. Samples were collected every 2
hours after the induction started, which lasted for 10 h. The
collected samples were treated for 10% SDS-PAGE analysis, and the
result was shown in FIG. 11.
As shown in the electrophorogram, the highest expression amount was
observed at 30.degree. C., with the shortest time used to achieve
the highest expression amount. The expression amount varied little
during 4 h-6 h. The expression amounts at both 25.degree. C. and
16.degree. C. were worse than that at 30.degree. C., with longer
induction time. Thus, the induction temperature and duration were
30.degree. C. and 5 h, respectively.
Based on the above results, the preferable process for fermentation
of the SpA5 engineering bacteria of the invention was finally
determined:
(1) animal origin TB medium was selected as the basal medium, with
the glycerol dosage of 10 mL/L;
(2) the inoculation amount was in a ratio of 10% at the beginning
of fermentation;
(3) the dissolved oxygen amount was maintained at around 45% all
the time during the course of fermentation;
(4) the induction was carried out at an IPTG concentration of 0.2
mM for 5 h at temperature of 30.degree. C.
2. Scale-Up of the Fermentation Process of Engineering Bacteria
Based on the above optimized conditions, the fermentation process
was scaled-up (25 L). The growth curve of the SpA5 engineering
bacteria (FIG. 12) and the electrophorogram of the expressed
proteins (FIG. 13) were obtained.
As shown in FIG. 12, a relatively standard growth curve was
observed after scaled-up, with a fast increasing curve at the
initial stage and a flat curve at the end stage of induction. The
bacterial density (OD.sub.600) reached 48 at the end of
fermentation, with a wet weight of 50 g/L.
As shown in FIG. 13, the expression amount of the target protein
increased significantly with extended time, and reached the maximal
amount at 5 h.
In conclusion, the scaling-up of the fermentation process was
successful for the SpA5 engineering bacteria, as expected. The
fermentation process of the present invention was suitable for
large-scale industrial production.
The engineering bacteria used in this Example were SpA5 (KKAA). The
fermentation process for other engineering bacteria was the same as
the engineering bacteria in this Example.
Example 4: Preparation and Purification of the SpA5 Protein
1. Supernatant Prepared from the Disrupted Bacteria
After fermentation, each recombinant engineering bacteria
constructed in Example 1 was collected by centrifugation. 200 g-500
g bacteria were mixed well and suspended in 20 mmol/L PB buffer (pH
7.0) in a ratio of 1:10 (w/v), and pre-cooled at 4.degree. C.
High pressure homogenization: after the pipeline of the high
pressure homogenizer (AH100B high pressure homogenizer, ATS
Industrial System Co. Ltd., Canada) was washed by distilled water,
cryogenic recycling system was started to pre-cool the equipment to
1-10.degree. C. for ready use. The pre-cooled bacteria suspension
was added to the high pressure homogenizer at a pressure maintained
at 730-770 Bar to disrupt the bacteria for 2-5 times. The disrupted
bacteria solution was smeared on a slide and stained by crystal
violet, then observed by an oil immersion lens. Less than 2
nondisrupted bacteria in each visual field were considered as
"complete disruption" (disruption rate >90%).
High speed centrifugation: after disruption, the suspension was
loaded into centrifugal barrels equipped for the large high-speed
centrifuge (Beckman, US), and centrifugated at 10,000-15,000 g for
15-30 min at 4.degree. C. After centrifugation, the supernatant was
collected.
2. Purification Using GST-Sepharose 4B Affinity Chromatography
To every 1 L supernatant, 200 mL GST media was added, allowing for
binding for more than 4 h at 4-25.degree. C. During this process,
vertical rotation or stirring was adopted to promote binding
between the target protein and the GST media. The target
protein-bound GST media was then washed by PBS for 5 volumes, to
remove the protein unbound to the GST-media. Subsequently, to every
100 mL GST media, 20 mL PP enzyme was added. The digestion was
carried out at 4-25.degree. C. for more than 2 h. After digestion,
the mixture was subjected to suction filtration and the filtrate
was collected. The target protein without GST-tag was obtained and
subjected to 12% SDS-PAGE analysis. The results were shown in FIG.
14. The result of purification of SpA5 (KKAA) was shown in FIG. 14,
which was similar to that for other SpA5 proteins.
3. Purification Using Cation-Exchange Chromatography
The theoretical isoelectric point of each recombinant SpA5 mutant
was larger than 8. Accordingly, conventional chromatography media
SP HP (strong cation-exchange media), MMC and Phenyl HP media were
firstly used for fine purification. Finally, Q HP was used to
remove endotoxin after fine purification of SpA5. Fine purification
process using chromatography includes:
1) Selection of Chromatography Medias
The purification processes of the SpA5 protein using SP HP, MMC and
Phenyl HP medias were compared. The sample used was the SpA5
protein obtained above after crude purification.
Instrument system: AKTA-explorer 100/Avant25 liquid chromatography
system (GE)
Medias for chromatography: SP HP, MMC, and Phenyl HP
Column sizes: (1) (.PHI.) 1.6 cm.times.(H) 2.5 cm, (2) (.PHI.) 2.6
cm.times.(H) 20 cm, and (3) (.PHI.) 1.6 cm.times.(H) 2.5 cm;
Packing volume: (1)(3) 5 mL.times.2, (2) 54 mL;
SP HP buffer: buffer A: 20 mM PB, pH 7.5; buffer B: 20 mM PB+1 M
NaCl, pH 7.5;
MMC buffer: buffer A: 20 mM Tris, pH 8.0; buffer B: 20 mM
Tris-HCl+1 M NaCl, pH 8.0;
Phenyl HP buffer: buffer A: 20 mM PB+1.5 M
(NH.sub.4).sub.2SO.sub.4, pH 6.0; buffer B: 20 mM PB, pH 6.0;
Loading samples: each SpA5 mutant protein after crude purification,
adjusted to the pH value identical to that of corresponding buffer
A for each media.
SP HP: loading flow rate: 8 mL/min, and elution flow rate: 8
mL/min;
Elution procedure: 0-30% buffer B for 10 column volumes (CV);
MMC: loading flow rate: 12 mL/min, and elution flow rate: 12
mL/min;
Elution procedure: 0-30% buffer B for 7 column volumes (CV);
Phenyl HP: loading flow rate: 8 mL/min, and elution flow rate: 8
mL/min;
Elution procedure: 0-100% buffer B for 10 column volumes (CV);
In each elution procedure, the rest fraction of buffer corresponded
to buffer A.
Collection: the target protein samples collected at each elution
step were subjected to SDS-PAGE analysis to evaluate the
purity.
FIG. 15-20 showed the purification results of SpA5 (KKAA). The
results for purifying other SpA5 proteins were consistent with that
for SpA5 (KKAA).
As shown in FIG. 15-20, higher purity of the target protein was
obtained by SP HP media than MMC and Phenyl HP media, which was
more than 90%, demonstrating that favorable purification could be
achieved by SP HP media. Based on the above reasons, SP HP media
was accordingly selected as media for the first step of
purification.
2) Optimization of the Purification Process by Chromatography
Based on determination of using SP HP media in the first
purification step, the conditions for purification, mainly
including: the conductivity of the loading sample and the elution
procedure were optimized in terms of the purity and the yield of
SpA5.
Instrument system: AKTA-explorer 100 liquid chromatography system
(GE Healthcare)
Media for chromatography: SP HP;
Column size: (.PHI.) 1.6 cm.times.(H) 2.5 cm;
Packing volume: 5 mL.times.2;
Buffer A: 20 mM PB, pH 7.5; buffer B: 20 mM PB+1 M NaCl, pH
7.5;
Loading samples: the SpA5 protein after crude purification,
adjusted to the pH 8.0;
Loading flow rate: 8 mL/min, and elution flow rate: 8 mL/min;
Elution procedure: (1) 0-100% B for 10 column volumes (CV); (2)
0-30% B for 10 column volumes (CV); The rest fraction in the eluent
was buffer A.
As shown in FIG. 21-22, no target protein was observed in the
flow-through fraction using the loading sample with low
conductivity, and the peak of target protein appeared very early.
While as shown in FIG. 23-24, no target protein was obviously seen
in the flow-through fraction with increased conductivity of the
loading sample. In order to increase the recovery of the target
protein, the sample was loaded at low conductivity. Based on the
above reasons, the conductivity of the loading sample was about 5
ms/cm, and the elution procedure was 0-50% B, 5 CV.
4. Purification by Ammonium Sulfate Precipitation
After purification by SP HP chromatography, the purity of SpA5 was
more than 90%. However, it was still demanded for further
purification to remove the band immediately below the target
protein as seen in the electrophoretogram. The sample was further
purified by ammonium sulfate precipitation to make SpA5
precipitated and leave the impurity protein in the supernatant.
Determination of the Conditions for Ammonium Sulfate
Precipitation:
The sample of SpA5 obtained after SP HP purification was mixed with
ammonium sulfate in various ratios, stirred, and centrifugated. The
supernatant and the precipitate were individually collected, and
subjected to SDS-PAGE for purity analysis.
Samples: the SpA5 protein after SP HP purification.
Ammonium sulfate: 3 M;
Conditions for precipitation: the sample was mixed with 3 M
(NH.sub.4).sub.2SO.sub.4 in different ratios, including: 1:2 and
1.4:1.6 (v/v) at 4.degree. C., followed by stirring for 10 min and
centrifugation at 6000 r/min for 20 min;
Result analysis: the supernatant and the precipitate after
centrifugation were individually collected and subjected to
SDS-PAGE to evaluate the removal of impurity protein by ammonium
sulfate precipitation at different concentrations (FIG. 25).
FIG. 25 showed the result of SpA5 (KKAA) purification by ammonium
sulfate precipitation. The results for the rest SpA5 proteins were
consistent with SpA5 (KKAA). As seen from the purity comparison
among proteins in FIG. 25, the removal result of impurity protein
by mixing the SpA5 (KKAA) sample after SP HP purification with 3 M
(NH.sub.4).sub.2SO.sub.4 in a ratio of 1.4:1.6 at 4.degree. C. is
the best, the purity of SpA5 was more than 95%. Accordingly, the
preferable precipitation conditions of the invention included:
mixing the SpA5 sample with 3 M (NH.sub.4).sub.2SO.sub.4 in a ratio
of 1.4:1.6 at 4.degree. C., followed by centrifugation and
collection of the precipitate of the SpA5 protein.
5. Desalinization
G25 desalinization column was equilibrated by the vaccine diluent,
then the sample obtained from the previous purification step was
loaded to the column to replace the buffer.
Specifically, the sample obtained from the previous purification
step was dissolved in the histidine buffer (histidine (Merck, US,
Pharmacuetical grade) 10 mmol/L, poloxamer 188 (Merck, US,
Pharmacuetical grade), 0.01% w/v, NaCl 9 g/L (Southwest
Pharmacuetical Co., Ltd.), and loaded to a pre-equilibrated XK
50-60 column (GE Healthcare, US) (600 mL Sephadex G 25) connected
to the chromatography system (AKTA Explorer 100, GE Healthcare,
US). The column was eluted at a flow rate of 20 mL/min for
desalinization (removal of (NH.sub.4).sub.2SO.sub.4 and PB).
6. Removal of Endotoxins
Instrument system: AKTA-explorer 100 liquid chromatography system
(GE Healthcare);
Media for chromatography: Q HP;
Column sizes: (.PHI.) 2.6 cm.times.(H) 20 cm;
Packing volume: 50 mL;
Buffers: buffer A: 10 mM His+0.01% poloxamer 188+0.9% NaCl, pH 6.0,
free of endotoxin (the vaccine diluent); buffer B: 1 M NaOH;
Loading samples: the sample obtained from the desalinization step,
adjusted to the pH value identical to buffer A for ready use.
The column was washed by buffer B (1 mol/L NaOH) in situ for 5
column volumes, and stood for 0.5 h. The system was then
equilibrated by the vaccine diluent to pH 6.0, followed by loading
the sample to the column. Flow rate: 8 mL/min. The flow-through
peak (i.e., the target protein) was collected.
The sample of the flow-through peak was subjected to 10% SDS-PAGE,
as shown in FIG. 26.
7. Scaling-Up
The process was scaled-up based on the conditions determined above:
the chromatography column of SP HP was scaled-up to (.PHI.) 2.6
cm.times.(H) 20 cm, with a packing volume CV=50 mL; the
chromatography column of Q HP was scaled-up to (.PHI.) 2.6
cm.times.(H) 20 cm, with a packing volume CV=50 mL. The experiment
was repeated for 3 times. The purity and the yield of SpA5 after
purification were analyzed by SDS-PAGE. The stability and the
repeatability of the process were evaluated after scaling-up.
As shown in FIG. 27-35, there was no obvious change between the
scaled-up process and the small-scale test for the purification of
the SpA5 protein by SP HP chromatography. After purification, the
purity of SpA5 was more than 95%.
8. Purity Detection by HPLC
HPLC instrument: Agilent 1260 (Agilent, US), analytical column:
ZorBax SB-300-C3 4.6.times.150 mm 3.5 micron (Agilent, US).
Mobile phases: A: 0.1% trifluoroacetic acid (Tedia, US), water
(18.2 M.OMEGA.); B: 0.1% trifluoroacetic acid (Tedia, US),
acetonitrile (Tedia, US).
Column temperature: 60.degree. C., flow rate: 0.5 mL/min, and
injection volume: 10 .mu.L.
Detection method: 0-30 min: 90% A, 10% B; 30-35 min: 100% B; 35-40
min: 90% A, 10% B; 40-45 min: 90% A, 10% B.
The results were shown in FIG. 36 and listed in Table 1.
As shown in FIG. 36 and Table 1, there was almost no impurity peak
in the sample of the purified SpA5 (KKAA) mutant. The retention
time of the main peak was 13.282 min, with the peak area ratio of
98.2%. Similar results were observed for the rest of mutants.
TABLE-US-00001 TABLE 1 Detection of peak by HPLC Peak Retention
time Peak width Peak Ratio of No. (min) Type (min) area (mAU * s)
Peak area 1 11.158 0.0000 0.00000 0.0000 2 12.879 BV 0.1387
26.23703 1.7774 3 13.282 VB 0.1186 1449.93225 98.2226 4 32.252
0.0000 0.00000 0.0000
9. N- and C-Terminal Sequencing, Molecular Weight Determination and
Amino Acid Composition Analysis of the Proteins
The resultant SpA5 mutants (including four SpA5 mutants of the
invention and SpA5ref (KKAA) were sequenced by Shanghai Applied
Protein Technology Co. Ltd. The results indicated that the
sequences of the mutants were consistent with the designed
sequences.
Example 5: Determination of the Endotoxin Content
1. The samples of Example 4 were diluted using water for bacterial
endotoxin detection (Zhanjiang Bokang Marine Biological Co., Ltd.)
to a concentration of 50 .mu.g/mL, and then used for detection. The
samples were diluted to 2 folds of the sensitivity (0.25 EU/mL) of
the endotoxin detection kit (Zhanjiang Bokang Marine Biological
Co., Ltd.).
2. According to Appendix XII E "Method for detection of bacterial
endotoxin" in "Pharmacopoeia of the People's Republic of China",
2010, the 3.sup.rd edition and the instructions of the kit,
positive control solution of endotoxin standards, working solution
of test sample, positive control solution of test sample, and
detection solution of test sample were prepared.
3. Preparation of Limulus Amoebocyte Lysate (LAL): based on the
amount of the test samples and the control samples, LAL was
prepared. Before opening the LAL vial, the bottleneck was
sterilized by alcohol swab and air-dried. To each vial, 0.1 mL
detection water was added, and gently mixed for further use.
4. Sample addition: to the prepared LAL, 0.1 mL of each of the
following solutions were added: detection solution of test sample,
positive control solution of test sample, positive control solution
of endotoxin standards and detection water. The mixture was gently
mixed, sealed by parafilm, and then placed in a water bath at
37.degree. C. for 60.+-.2 min, during this period, the samples
should not be moved. Detection water was used as a negative
control.
5. Detection: LAL was taken out from the water bath carefully, and
gently inverted vertically to observe the bottom. Intact gel that
did not slide along the wall of test tube was considered as
positive, and designated as (+); while broken gel that slided along
the wall of test tube was considered as negative, and designated as
(-).
6. Results: all mutant protein solutions were negative, with
endotoxin content less than 5 EU/ml.
Example 6: Binding Between Each Coated Protein and Human IgG
The resultant proteins were each diluted by 0.1 mol/L carbonate
buffer (pH 9.5) to a concentration of 500 ng/ml. A 96-well ELISA
plate was then coated by 100 .mu.L of the diluted protein solution
at 4.degree. C. over night. The plate was washed by the washing
solution (Tris 2.42 g, Tween 20 0.5 mL, adjusted to pH=7.4 by
concentrated HCl, adding ddH.sub.2O to a total volume of 1000 mL)
for 4 times. To each well, 200 .mu.L blocking solution (5% (w/v)
skimmed milk powder dissolved in 100 mmol/L TTBS (Tris (pH 7.5),
Tween 20 0.1% (v/v), and NaCl 0.9% (w/v)) was added for blocking at
37.degree. C. for 2 h. The liquid was discarded, and the plate was
washed for 4 times by the washing solution. 100 .mu.L human IgG-HRP
(Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd.) (1:5000)
was added and incubated at 37.degree. C. for 60 min. The liquid was
discarded, and the plate was washed for 4 times by the washing
solution. 100 .mu.L/well freshly prepared developing solution
containing OPD was added, and developed at room temperature in dark
for 15 min. 50 .mu.L 12.5% H.sub.2SO.sub.4 was added to stop the
reaction. OD.sub.450nm was then detected. Higher OD value indicated
stronger binding to human IgG, further suggesting that it was not
suitable as an antigen candidate. Results were shown in FIG. 37 and
listed in Table 2, in which PBS was used as a negative control.
TABLE-US-00002 TABLE 2 Binding capacities of SpA and each mutant
protein thereof to human IgG detected by ELISA SpA5 SpA5Ref SpA5
SpA5 SpA5 SpA5 Groups PBS SpA wt (KKAA) (KKAA) (KKVV) (RRAA) (RRVV)
OD.sub.450 nm 0.05 0.629 0.615 0.252 0.166 0.217 0.189 0.027 0.068
0.611 0.618 0.187 0.231 0.173 0.179 0.1 0.047 0.65 0.618 0.242
0.209 0.167 0.2 0.07 0.08 0.665 0.579 0.186 0.21 0.128 0.19 0.036
0.065 0.675 0.622 0.198 0.197 0.177 0.226 0.061 0.074 0.623 0.668
0.231 0.189 0.19 0.226 0.062 0.05 0.639 0.689 0.212 0.201 0.173
0.207 0.055 0.049 0.56 0.629 0.194 0.16 0.184 0.174 0.084 0.019
0.554 0.621 0.19 0.203 0.118 0.196 0.069 0.092 0.663 0.587 0.228
0.206 0.164 0.264 0.033 0.051 0.688 0.647 0.199 0.169 0.235 0.185
0.06 0.092 0.661 0.61 0.216 0.231 0.174 0.205 0.088 Mean 0.061
0.635 0.625 0.211 0.198 0.175 0.203 0.062 value Standard 0.021
0.043 0.031 0.023 0.023 0.032 0.025 0.022 deviation Paired 0.208
<0.01 0.335 <0.01 t-test (compared with SpA5ref (KKAA) P
As indicated by the results, binding capacities to human IgG of
each SpA5 protein of the invention decreased significantly as
compared to the wild-type SpA5wt and SpA (252), P<0.01; while
SpA5 (RRVV) had already lost its binding capacity to human IgG.
Example 7: Binding Between SpA and Human IgG Blocked by Rabbit
Anti-Recombinant Mutant Antibody F(ab).sub.2 Fragments
Based on the results of Example 6, SpA5wt had the same binding
capacity to human IgG as the intact SpA protein, so that both
proteins were not subjected to this experiment.
1) Immunization of Rabbit
1 mL complete Freund's adjuvant (Sigma, US) was added to each of 2
mg SpA5ref (KKAA), SpA5 (KKAA), SpA5 (KKVV), SpA5 (RRAA) and SpA5
(RRVV) (2 mg/ml), and the mixture was fully emulsified. 25 New
Zealand big ear rabbits (2.0-2.5 kg, Laboratory Animal Centre,
Third Military Medical University) were divided into 5 groups, with
5 rabbits in each group. The rabbits were subcutaneously immunized
at multi-site by 2 mL emulsified protein (protein content of 2 mg)
on Day 0, Day 14 and Day 28. Serum was collected on the Day 7 after
the last immunization.
2) Purification of the Antibodies
After ammonium sulfate precipitation, the rabbit serum was diluted
by PBS, and subsequently loaded to a pre-packed Protein G column
(GE Healthcare, US) pre-equilibrated by PBS. The column was then
washed by PBS, and eluted by a buffer containing 1 mol/L glycine
and 0.5 mol/L NaCl (pH 2.5). The eluent was immediately neutralized
by 1 mol/L Tris-HCl buffer (pH 8.5) and dialyzed against PBS. The
antibody concentration was determined as 3 mg/ml by Lowry method.
The antibody was an SpA5 specific antibody, as confirmed by western
blot.
3) Preparation of Antibody F(ab).sub.2 Fragments
A 60 IU/ml pepsin (Sigma, US) solution was formulated using an
acetate buffer (sodium acetate 18 g, glacial acetic acid 9.8 mL,
adding water to dilute to a volume of 1000 mL, pH 4.5). The pepsin
solution was then added to the antibodies obtained in the previous
step in a ratio of 1:11 (v/v), and maintained at 37.degree. C. for
30 min. The reaction was stopped by adding 1 mol/L Tris-HCl buffer
(pH 8.5). Purification by affinity chromatography was conducted
using a pre-packed Protein G column as described above. The
concentration was 1 mg/ml as detected by Lowry method. The results
were shown in FIG. 38.
4) Verification of the Binding Between SpA and Human IgG Blocked by
Anti-Recombinant Mutant Antibody F(ab).sub.2 Fragments
The SpA5 protein was each diluted by 0.1 mol/L carbonate buffer (pH
9.5) to a concentration of 500 ng/ml. A 96-well ELISA plate was
coated by the diluted protein solutions at 4.degree. C. over night.
The plate was then washed by the washing solution (Tris 2.42 g,
Tween 20 0.5 mL, adjusted to pH=7.4 by concentrated HCl, adding
ddH.sub.2O to a total volume of 1000 mL) for 4 times. To each well,
200 .mu.L blocking solution (5% (w/v) skimmed milk powder dissolved
in 100 mmol/L TTBS (Tris (pH 7.5), Tween 20 0.1% (v/v), and NaCl
0.9% (w/v)) was added for blocking at 37.degree. C. for 2 h. The
liquid was discarded, and the plate was washed for 4 times by the
washing solution. 100 .mu.L human IgG-HRP (Beijing Zhongshan Golden
Bridge Biotechnology Co. Ltd., Beijing) (1:5000) and 100 .mu.L
rabbit anti-mutant protein antibody F(ab).sub.2 fragments diluted
to a concentration of 100 ng/ml were added to react at 37.degree.
C. for 60 min. The liquid was discarded, and the plate was washed
for 4 times by the washing solution. 100 .mu.L freshly prepared
OPD-containing developing solution was added per well, and
developed at room temperature in dark for 15 min. 50 .mu.L 12.5%
H.sub.2SO.sub.4 was then added to stop the reaction. OD.sub.450nm
was detected. Higher OD value indicated stronger binding to human
IgG further suggesting that it was not suitable as an antigen
candidate. The results were listed in table 3 and shown in FIG.
39.
TABLE-US-00003 TABLE 3 The ELISA results of binding between SpA and
human IgG blocked by anti-recombinant mutant protein antibody
F(ab).sub.2 fragments (OD.sub.450 nm) Paired t-test (compared with
SpA5ref (KKAA) Ave stdev control) P PBS 0.636 0.665 0.601 0.698
0.605 0.641 0.041 Negative rabbit 0.593 0.547 0.558 0.497 0.568
0.553 0.035 serum F(ab).sub.2 Anti-SpA5ref 0.289 0.363 0.293 0.345
0.313 0.321 0.032 -- (KKAA) antibody F(ab).sub.2 Anti-SpA5 0.226
0.305 0.244 0.293 0.267 0.267 0.033 <0.01 (KKAA) antibody
F(ab).sub.2 Anti-SpA5 0.351 0.320 0.343 0.342 0.299 0.331 0.021
0.628 (KKVV)antibody F(ab).sub.2 Anti-SpA5 0.247 0.204 0.243 0.273
0.213 0.236 0.028 <0.05 (RRAA) antibody F(ab).sub.2 Anti-SpA5
0.102 0.068 0.075 0.115 0.067 0.085 0.022 <0.01 (RRVV) antibody
F(ab).sub.2
As indicated by the results, the antibodies generated from each
SpA5 protein of the invention would effectively bind to SpA, so
that the binding capacity of the SpA to human IgG was reduced
significantly, in which anti-SpA5 (RRVV) antibody F(ab).sub.2
reduced the most.
Example 8: Inductive Apoptosis of B Cells
1) 64 BALB/c mice (6-week old, body weight of about 16 g, Beijing
HFK Bioscience Co., Ltd.) were randomly divided into 7 groups, with
8 mice in each group. The groups were designated as PBS group, SpA
group, SpA5wt group and each mutant group.
2) For SpA group, purified SpA was injected I.P. at a dosage of 150
.mu.g/mouse; for SpA5wt group, purified wild-type SpA5wt protein
was injected I.P. at a dosage of 150 .mu.g/mouse; for SpA5 mutant
group, the purified SpA5 mutant protein was injected I.P. at a
dosage of 150 .mu.g/mouse; for PBS group, equal volume of PBS was
injected.
3) 4 h later, mice were killed by breaking the neck. The spleens
were taken from the mice in each group, and PBS was added. Single
cell suspension was then prepared by a 200-mesh screen.
Subsequently, the suspension was centrifugated at 1000 rpm for 5
min, and the supernatant was discarded.
4) Erythrocyte lysis solution (BD Biosciences, US) at 4.degree. C.
was added to the cell precipitate in a ratio of 1:5 (i.e. 5 mL
lysis solution was added to 1 mL cell). The mixture was gently
pipetted to homogeneity, and stood at room temperature for 5 min.
Subsequently, it was centrifugated at 800-1000 rpm for 5 min, and
the red supernatant was discarded and precipitate was then
collected, to which Hank's solution (Hyclone, US) or serum-free
medium 1640 (Hyclone, US) was added, followed by washing via
centrifugation for 3 times. Complete medium 1640 (Hyclone, US) was
then added to adjust the cell density to 2.times.10.sup.6
cell/ml.
5) Cell collection: cells were collected directly to an U-shaped
96-well plate. Centrifugation: the U-shaped plate was centrifugated
directly on an U-shaped plate rack at 1800 rpm for 5 min. After
centrifugation, the supernatant was discarded, and the precipitate
was re-suspended by 160 .mu.L staining buffer (PBS+1% fetal bovine
serum (GIBCO New Zealand). The suspension was then centrifugated in
the same manner as described above; centrifugated in a 1.5 mL Ep
tube at 1800 rpm for 5 min in a small centrifuge. After the
supernatant was discarded, the precipitate was re-suspended by 1 mL
staining buffer, and then centrifugated in the same manner as
described above.
6) Flow cytometry staining: during centrifugation, the staining
solution of recombinant phycoerythrin coupled anti-mouse CD19
antibody (eBioscience, US) was pre-prepared, and subsequently,
diluted to 2-fold dilution as required. The solution was then added
at 50 .mu.L/well to the centrifugated wells above, and incubated at
4.degree. C. for 30 min. The precipitate was slightly re-suspended
by adding 100 .mu.L staining buffer, and the mixture was
centrifugated at 1800 rpm for 5 min. After the supernatant was
discarded, 160 .mu.L staining buffer was added for re-suspension.
The mixture was then centrifugated at 1800 rpm for 5 min.
7) Detection by a flow cytometer: after the supernatant was
discarded, the precipitate was re-suspended by adding 150 .mu.L
1.times.PBS, and subsequently placed into a detection tube. Flow
cytometry BD FACSCanto II was performed according to the
procedure.
The closer percentage of CD19+ leucocytes to that of the negative
control indicated a weaker apoptosis-inducing capacity of the test
protein, so that it was more suitable as an antigen candidate. The
results were listed in Table 4 and shown in FIG. 40.
TABLE-US-00004 TABLE 4 Percentage of CD19+ B-cells Paired t-test
(compared with SpA5ref Mean stdev (KKAA)control) P PBS 54 51 52 48
56 51 50 54 52 2.6 SpA(252) 40 36 42 43 35 43 34 39 39 3.6 SpA5wt
33 39 33 39 34 39 31 40 36 3.6 SpA5ref(KKAA) 36 48 40 44 39 45 39
45 42 4.1 -- SpA5(KKAA) 42 52 53 41 47 46 48 47 47 4.2 <0.05
SpA5(KKVV) 41 47 49 38 39 45 42 43 43 3.8 0.558 SpA5(RRAA) 44 53 50
48 51 53 45 48 49 3.4 <0.01 SpA5(RRVV) 54 52 47 53 47 53 48 54
51 3.1 <0.01
As indicated by the results, apoptosis of mouse spleen B
lymphocytes could be induced by both SpA (252) and SpA5wt, whereas
for all mutant proteins, apoptosis could not be induced
(Mean.+-.stdev, n=8).
Based on the Examples above, the expression amount and the binding
capacity to human IgG of each SpA5 protein of the invention, the
capacity of the anti-SpA5 mutant antibody F(ab).sub.2 fragment to
block the binding between SpA and human IgG and their B-cell
apoptosis-inducing capacity were compared with those of SpA5ref
(KKAA). The results were listed in Table 5.
TABLE-US-00005 TABLE 5 Summary of the properties of each SpA5
mutant SpA5 SpA5 SpA5 SpA5 (KKAA) (KKVV) (RRAA) (RRVV) Expression
amount No No Relatively Relatively lower difference difference
lower Binding capacity to No lower No difference Very significantly
human IgG difference lower Capacity to block binding Very No
Significantly Very significantly between SpA and human
significantly difference higher higher IgG by antibody F(ab).sub.2
higher fragment B-cell apoptosis-inducing Significantly No Very
Very significantly capacity lower difference significantly lower
lower
Example 9: Preparation of the SpA5 Vaccine
Aluminum phosphate adjuvant was an imported product with original
packaging from GENERAL CHEMICAL corporation (US) (concentration of
element aluminium: 5.3-5.4 mg/ml);
1. Preparation of Staphylococcus aureus Recombinant SpA5
Vaccine
(1) Exact 800 .mu.L aluminum phosphate adjuvant was measured out
and placed into a formulation bottle. Subsequently, 2200 .mu.L
vaccine diluent (histidine 10 mM, NaCl 0.9%, poloxamer 188 0.01%,
pH 6.0) was measured out and added to the bottle to a final volume
of 3000 .mu.L. The solution was then fully mixed.
(2) Each SpA5 protein was diluted to a concentration of 300
.mu.g/3000 .mu.L by the vaccine diluent, and fully mixed;
(3) The diluted adjuvant solution was added with the diluted
protein solution of the same volume to a formulation bottle. The
mixture was then suspended vertically or stirred horizontally at
the temperature in the range from 4 to 37.degree. C. After
adsorption for 1 h, the vaccine was prepared.
2. Adsorption Homogeneity and Completeness of the Recombinant
Antigen Proteins in the Staphylococcus aureus Vaccine by the
Aluminum Phosphate Adjuvant as Characterized by 12% SDS-PAGE
(1) 1 mL vaccine formulation as described above was centrifugated
at 6000 rpm for 5 min at 4.degree. C. The supernatant after
centrifugation was carefully taken out, from which 40 .mu.L sample
was withdrawn.
(2) The dissociation solution (1 M Na.sub.2CO.sub.3) was added of
the same volume as the supernatant withdrawn, and suspended
vertically at room temperature for 1 h. Subsequently, 40 .mu.L
suspension was sampled.
(3) Protein solution free of the aluminum phosphate adjuvant was
prepared according to the method described above in 1, in which the
volume of the aluminum phosphate solution was substituted by the
vaccine diluent. After fully mixed, 40 .mu.L sample was
collected.
(4) To the sample collected, 10 .mu.L 5.times. loading buffer was
added. The mixture was then heated at 100.degree. C. for 5 min.
After cooling down, the samples were centrifugated instantly. 10
.mu.L samples were loaded to the gel.
(5) The procedure for 12% SDS-PAGE included initially running at 80
v for 20 min, and then at 180 v for 40 min. After running, the gel
was stained in a Coomassie staining solution under shaking,
followed by destaining in a destaining solution under shaking. The
gel was observed in an imaging system. The results were shown in
FIG. 41, which indicated that the proteins could be fully adsorbed
by the aluminum phosphate adjuvant.
Example 10: Establishment of a Standard Curve for Quantitative
Analysis of the Staphylococcus aureus Strain (International
Standard Strain MRSA-252) Used for Infection
The strain was inoculated to an MH agar plate, and incubated at
37.degree. C. for 24 h. Single colony was individually picked up
from the plate, inoculated to MH liquid medium, and incubated at
37.degree. C. in a shaker for 6 h. Subsequently, bacteria were
collected by centrifugation at 6000 rpm for 10 min, and washed
twice by physiological saline. The bacteria solution was diluted to
10- and 1.25-fold, respectively. The absorbance at 600 nm
(OD.sub.600) was detected by an ultraviolet spectrometry for each
bacteria solution. 100 .mu.L diluted bacteria solution was smeared
on an MH agar plate, and incubated at 37.degree. C. for 24 h,
followed by colony counting. Based on the colony numbers on each
plate and the OD.sub.600 values for bacteria solution, a standard
curve was established.
Results: the equation for the standard curve was Y=2.3065X+0.0051
(10.sup.9 CFU/ml), with a correlation coefficient of 0.9999.
Example 11: Establishment of a Septicopyemia Animal Model
1. MRSA-252 was inoculated to an MH agar plate, and incubated at
37.degree. C. for 24 h. Single colony was individually picked up
from the plate, inoculated to MH liquid medium, and incubated at
37.degree. C. in a shaker for 6 h and the bacteria were collected.
Based on the standard curve, the bacteria were quantified, and the
bacteria solution was diluted (or concentrated) to various
concentrations, including 2.0.times.10.sup.10 CFU/mL,
1.5.times.10.sup.10 CFU/mL, 1.25.times.10.sup.10 CFU/mL, and
1.0.times.10.sup.10 CFU/mL. Subsequently, BALB/C mice (6-8 weeks
old, body weight of 18-20 g) were systemically infected by the
bacteria solutions of various concentrations through tail
intravenous injection (100 .mu.L/animal). The physiological saline
was used as a control. After observed for 7 days, the death rate
was determined for each group.
2. The amount of colonized bacteria was detected by colony counting
every 24 h after infection (until Day 7 after infection): 3 mice
were randomly selected from each of the infection groups and
control group, from which 0.5-1 mL blood sample was taken by
eyeball enucleation. 20 .mu.L blood sample was diluted 10 folds in
180 .mu.L heparin for bacteria counting. 50 .mu.L sample was
smeared on an agar plate, and incubated at 37.degree. C. for 24 h
for clone counting. The mice were killed after blood sample
collection. After sterilization by soaking in 75% aqueous alcohol
solution, the limbs of mice were fixed. Subsequently, the mice was
dissected and the spleen, kidney, and liver were taken out, and
placed in 2 mL sterile PBS. The organs were homogenized in a clean
glass homogenizer, and 1 mL homogenate was each diluted in a ratio
of 1:10, 1:100, or 1:1000. 100 .mu.L of each dilution was gently
smeared on solid medium, and incubated at 37.degree. C. for 24 h
for colony counting. The results were listed in Table 6.
TABLE-US-00006 TABLE 6 Minimum lethal dose and sub-lethal dose of
MRSA-252 Total MRSA-252 number Mortality dose for Number of Number
of the mice died at various times of dead rate infection mice 12 h
24 h 48 h 72 h 96 d 120 d 6 d 7 d mice (%) 2.0 .times. 10.sup.9 CFU
30 30 0 0 0 0 0 0 0 30 100 1.5 .times. 10.sup.9 CFU 30 0 3 24 3 0 0
0 0 30 100 1.25 .times. 10.sup.9 CFU 30 0 0 24 0 3 3 0 0 30 100 1.0
.times. 10.sup.9 CFU 30 0 0 3 3 6 9 0 0 21 70
In the dosage group of 2.0.times.10.sup.9 CFU, the mortality rate
was 100% within 12 hours (h); in the dosage group of
1.5.times.10.sup.9 CFU, the mortality rate was 90% within 48 h, and
100% within 72 h; in the dosage group of 1.25.times.10.sup.9 CFU,
the mortality rate was 80% within 48 h, 90% within 96 h, and 100%
within 120 h; in the dosage group of 1.0.times.10.sup.9 CFU, the
mortality rate was 10% within 48 h, 20% within 72 h, and 70% with
in 7 days (d). Accordingly, the minimum lethal dose of MRSA-252 was
1.25.times.10.sup.9 CFU, and the sub-lethal dose was
1.0-1.25.times.10.sup.9 CFU.
3. Colonization counts in blood and various organs in the BALB/C
mouse infected by MRSA-252
A peak value of bacteria was attained in blood at 48 h after
infection, with maximal colonization counts of 8.0.times.10.sup.9
CFU/ml. The amount of bacteria in blood started to decrease from 72
h, and no bacterium was detected at 96 h. The peak values of
bacteria colonized in spleen, kidney, and liver were all reached at
72 h after infection, with maximal colonization counts of
8.0.times.10.sup.9 CFU/ml. In control group, the colonization
counts in blood, spleen, kidney, and liver were all 0.
In the results above, the survival rate of mice and colonization
counts of bacteria in blood, and major organs, including spleen,
kidney, and liver were evaluated in an animal model, which provided
a basis for successful development of single subunit vaccine and
multi-subunit fusion vaccine of SA, and for researches on the
pathogenesis of SA infections.
Example 12: The Effect of SpA5 as an Antigen Determined by Immune
Protection Against Challenging in Animals
1. According to the method of Example 9, SpA5ref (KKAA), SpA5
(KKAA) and SpA5 (RRVV) were prepared into vaccines.
2. Experiment animals and group division
Female BALB/C mice of 6-week old were used (Beijing HFK Bioscience
Co., Ltd.). The animals were divided into 5 groups, including
vaccine diluent group, aluminum phosphate adjuvant control group,
SpA5ref (KKAA) group, SpA5 (KKAA) group and SpA5 (RRVV) group, with
30 mice in each group.
3. Immunization was performed by quadriceps femoris injection of
each vaccine for 3 times (Day 0, 14, and 21) at a dosage of 30
.mu.g/100 .mu.L.
4. The procedure of challenging: after the last immunization,
viable MRSA-252 was injected via caudal vein at a lethal dose on
Day 14 for challenging test. The amount of bacteria suspension was
1.25.times.10.sup.9 CFU (determined based on the results listed in
Table 6) for each BALB/C mouse. After observed for 10 days, the
survival rate was calculated for each group.
5. The results were listed in Table 7.
TABLE-US-00007 TABLE 7 Protective capacity against challenging
after immunization by each SpA5 mutant protein Number of survival
animals after Groups 10 days Survival rate Vaccine diluent 4 13%
AlPO.sub.4 6 20% SpA5(KKAA) 11 37% SpA5(RRVV) 12 40% SpA5ref(KKAA)
10 33%
As indicated by the results listed in the Table above, favorable
protective effects of SpA5 (KKAA) and SpA5 (RRVV) were observed for
the animals.
B. Method for Detecting SpA5-Specific IgG
The SpA5 protein of the invention had strong immunogenicity, and
could used as an antigen candidate for the Staphylococcus aureus
vaccine. However, SpA5 was still able to partially bind the Fc
fragment of mammalian IgG resulting in difficulties in detecting
SpA5 antigen specific IgG As indicated by previous results of serum
detection in the animals immunized by SpA5, the inventors found
that the plate coated by the mutant SpA5 protein reacted with the
serum in negative control group, and presented a weakly positive
result, due to non-specific binding between SpA5 and the Fc
fragment of IgG in the serum.
On such a basis, it was intended to provide a detection method for
SpA5 specific antibody without non-specific interference.
Some parts of peptide chains of the immunoglobulin could be easily
hydrolyzed by proteases into different fragments. As one of the
most used proteolytic enzymes, pepsin was used to hydrolyze IgG
into one F(ab').sub.2 fragment and some small fragments pFc'.
F(ab').sub.2 was a divalent structure composed of 2 Fab fragments
and a hinge region, which retained its biological activity of
binding to corresponding antigens, and avoided the side effect of
the immunogenicity of Fc fragment. After hydrolysis by pepsin, pFc'
was finally degraded and lost its biological activity. Based on the
techniques above, the SpA5 antigen specific IgG antibodies were
detected in the serum of different animals immunized by the
recombinant Staphylococcus aureus vaccines. At present, no related
research has been reported yet.
In this section, the present invention provided a method for
detecting the SpA5 antigen specific IgG antibody. Using this
method, non-specific binding between SpA5 and the Fc fragment of
IgG antibody was avoided, so that the SpA5 antigen specific IgG
antibodies could be detected in various species after immunization
by the vaccines. This method provided a basis for researches on the
antigenicity, immunogenicity and immunoprotection of SpA5.
In the first aspect, the method for detecting the SpA5 antigen
specific IgG antibody of the invention comprised: 1) obtaining the
serum of the animal immunized by the SpA5 protein, and digesting
the antibody in the serum by pepsin to obtain F(ab').sub.2
fragments of the antibody; 2) detecting the SpA5 specific
F(ab').sub.2 fragment IgG antibody by ELISA.
Specific steps of the method were as follows:
1) Antibody preparation: the blood samples collected after
immunization of animals by the SpA5 protein were placed at
4.degree. C. for 2 h, and then centrifugated at 8000 rpm/min for 10
min at 4.degree. C. The supernatant serum was withdrawn, and stored
at -20.degree. C. for further use.
2) Preparation of the digestion solution: to 0.1-0.2 M sodium
acetate solution, pepsin was added to a final activity of 30-150
IU/mL, and preferably, 60 IU/mL. The pH value was adjusted to
4.0-4.6.
3) the serum obtained in step 1) was diluted 10 folds by the
digestion solution prepared in step 2). After fully mixed,
digestion was performed in a water bath at 37.degree. C. for 6 h.
During digestion, the mixture was shaken to mix well for 5-10 min
at an interval of 1 h. In this step, an F(ab').sub.2 fragment and
some small fragments pFc' were obtained after digestion of antibody
by pepsin, avoiding the interference of the Fc fragment.
4) SpA5 specific F(ab').sub.2 fragment IgG antibody titer was
detected by ELISA using the purified SpA5 protein; preferably, an
ELISA plate was coated by the SpA5 protein at a concentration of 2
.mu.g/ml. Subsequently, after the digested sample in step 3) was
diluted by the antibody diluent in a ratio of 1:2000, the SpA5
specific F(ab').sub.2 fragment IgG antibody titer was detected by a
series of fold-dilutions.
In another aspect, the present invention provides a kit for
detection of the SpA5 antigen specific IgG antibody, which
comprises: enzyme digestion buffer, pepsin, and reagents for ELISA.
Preferably, the enzyme digestion buffer was 0.1-0.2 M sodium
acetate solution; and preferably, the reagents for ELISA includes:
coating solution, antibody diluent, washing solution, blocking
solution and stopping solution. The reagents required for ELISA
could easily determined by the one skilled in the art based on
common skills in the field.
In the present invention, in order to obtain the antibody titer in
the serum, the SpA5 specific IgG antibody in the serum of the
animal immunized by the SpA5 vaccine was detected using digestion
by pepsin and ELISA test. In this method, the specificity was
enhanced by excluding the interference of non-specific binding
between SpA and the Fc fragment of antibody. Meanwhile, the method
had advantages of simple operation and good repeatability.
Additionally, the method was verified by the antibody detection in
the serums among various species (BALB/C mouse, SD rat, and New
Zealand big ear white rabbit). The method could be used for
researches on immunogenicity and antigenicity of the recombinant
Staphylococcus aureus vaccines.
The materials and primary reagents used in this section were as
follows:
1. Experiment Animals
BALB/C mice (Beijing HFK Bioscience Co., Ltd.), SD rats (Beijing
Charles River Laboratories. Inc.) and New Zealand big ear white
rabbits (Laboratory Animal Center, Third Military Medical
University).
2. Materials
SpA5 (KKAA) (30 .mu.g/600 .mu.L) vaccine formulation.
3. Primary Reagents
Glycine and pepsin were purchased from Shanghai Sangon Biotech.
Inc. Ordinary and F(ab').sub.2 fragment goat anti-mouse, rat and
rabbit IgG secondary antibodies were supplied by Shanghai
Hengdailao Commerce Co., Ltd. Soluble substrate solutions of
individual component were obtained from an agent in Chongqing of
Tiangen Biotech (Beijing) Co., Ltd. Sodium chloride, potassium
dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate,
and Polysorbate 20 were obtained from Sinopharm Chemical Reagent
Co. Ltd. Potassium chloride was purchased from Chengdu Kelong
Chemical Reagent Factory. PBS buffer was from Beijing Zhongshan
Golden Bridge Biotechnology Co. Ltd.
4. Preparation of Reagents
1) enzyme digestion solution: to 0.1-0.2 M sodium acetate solution,
pepsin was added to a final activity of 60 IU/mL, and the pH value
was adjusted to 4.0-4.6.
2) Reagents for ELISA
{circle around (1)} Coating solution: 1.6 g Na.sub.2CO.sub.3 and
2.9 g NaHCO.sub.3 were weighed on a electronic balance, and the pH
value was adjusted to 9.6, distilled water was added to a final
volume of 1000 mL.
{circle around (2)} Antibody diluent: 8 g NaCl, 0.2 g
KH.sub.2PO.sub.4, 2.9 g Na.sub.2HPO.sub.4.12H.sub.2O, 0.2 g KCl
were weighed on a electronic balance, 0.5 mL Tween 20, and the pH
value was adjusted to 7.4, distilled water was added to a final
volume of 1000 mL.
{circle around (3)} Washing solution: 0.05% Tween 20-PBS (pH
7.4)
1 bag of 1000 mL/bag PBS was dissolved in 1000 mL pure water,
followed by adding 0.5 mL Tween 20.
{circle around (4)} Blocking solution (freshly prepared)
BSA was added in a ratio of 1% to 20 mL antibody diluent, and
stored at 4.degree. C. for further use.
{circle around (5)} Stopping solution (2 mol/L sulphuric acid
solution)
22.2 mL concentrated sulfuric acid was added to 177.8 mL
ddH.sub.2O.
Example 13: Animal Immunization and Preparation of Immunized
Serum
1) Group division of animals: the animals were randomly divided
into immunization group by vaccine and control group. The groups
were listed in Table 8.
TABLE-US-00008 TABLE 8 Group division of experiment animals for
immunogenicity investigation Immunization Control Animal species
group by vaccine group BALB/C mouse 10 10 SD rat 5 5 New Zealand
big ear white 2 2 rabbit
2) Immunization of the experiment animals: for immunization group,
the animals were immunized by intramuscular injection of a dose of
SpA5 (KKAA) (600 .mu.L) per animal; for control group,
physiological saline of the same volume was used. The procedure
included immunization for 3 times on Day 0, Day 14 and Day 21.
3) Blood collection: blood was collected on Day 14 after the last
immunization. The blood sample was collected by eyeball enucleation
for mice, via caudal vein for rats, and via auricular vein for
rabbits. The blood was incubated at 4.degree. C. for 2 h, and then
centrifugated at 8000 rpm for 10 min. Subsequently, the serum was
separated and stored at -20.degree. C. for further use.
Example 14: Digestion of Antibodies in the Serum by Pepsin
20 .mu.L serum obtained in Example 13 was added to 180 .mu.L enzyme
digestion solution. After fully mixed, the serum was digested in a
water bath at 37.degree. C. for 6 h. During digestion, the mixture
was shaken to mix well for 5 min at an interval of 1 h.
Example 15: Detection of Specific Anti-SpA5 (KKAA) Antibody
Methods
1) Coating: purified SpA5 (KKAA) protein was diluted to 2 .mu.g/mL
by the coating solution. An ELISA plate was then coated at a
concentration of 100 .mu.L/well. After fully shaken for
homogeneity, the plate was placed in a refrigerator at 4.degree. C.
overnight or at 37.degree. C. for 2 h.
2) Blocking: the plate was washed by the washing solution (4 times,
each 300 .mu.L). For each washing, the plate was shaken for 30 s
and the washing solution was pipetted for 2.5 s. The ELISA strip
was blocked by the blocking solution at 200 .mu.L/well, and placed
in a refrigerator at 4.degree. C. overnight or at 37.degree. C. for
2 h.
3) Addition of the primary antibody: the plate was washed by the
washing solution (4 times, each 300 .mu.L). For each washing, the
plate was shaken for 30 s and the washing solution was pipetted for
2.5 s. The serum digested above was diluted by 200 folds to 1:2000.
Subsequently, a series of 7 2-fold dilutions were obtained using
the antibody diluent. The resulting dilutions were homogenously
mixed by shaking. The diluted samples were added to an ELISA plate
at 100 .mu.L/well, and incubated at 37.degree. C. for 40 min.
4) Addition of the secondary antibody: the plate was washed by the
washing solution (4 times, each 300 .mu.L). For each washing, the
plate was shaken for 30 s and the washing solution was pipetted for
2.5 s. Goat anti-mouse, rat and rabbit IgG F(ab').sub.2 fragment
secondary antibody labeled by HRP was diluted by the antibody
diluent to 1:10000. The dilutions were added at 100 .mu.L/well to
the plate, homogenously mixed by shaking, and incubated at
37.degree. C. for 40 min. Meanwhile, common goat anti-mouse, rat
and rabbit IgG secondary antibodies were used as a control.
5) Developing: the plate was washed by the washing solution (4
times, each 300 .mu.L). For each washing, the plate was shaken for
30 s and the washing solution was pipetted for 2.5 s. The
developing solution was added at 100 .mu.L/well. The mixture was
then developed in dark for 5-10 min.
6) Termination of the reaction: after developing, 2 mol/L
H.sub.2SO.sub.4 was added at 50 .mu.L/well to stop the reaction. OD
values for each well were detected at 492 nm by a micro-plate
reader.
7) Statistical analysis: A.sub.sample/A.sub.negative>2.1 was
regarded as positive standard. The maximal dilution was determined
for each specific antibody in the serum. A histogram was plotted
using the geometric mean titer.
Results:
As shown in Table 9, the ELISA results indicated that: the results
of immunization by vaccine group and control group are similar when
a common IgG secondary antibody was used in the detection, since
SpA5 (KKAA) comprised an IgG binding domain, which non-specifically
bound to the Fc fragment of the mammalian IgG antibody, there was
no significant difference. When goat anti-mouse, rat and rabbit IgG
F(ab').sub.2 fragment secondary antibody was used, negative results
were observed for all samples in control group, so that
non-specific interference was avoided. By using this method, the
actual level of SpA5 (KKAA) specific IgG antibody in the serum can
be detected more precisely, with higher specificity. In order to
analyze the results, a histogram was plotted using the geometric
mean titer (GMT) of the antibody detected in the serum of mice in
individual experiment groups of various species, as shown in FIG.
42.
TABLE-US-00009 TABLE 9 Detection of the specific antibody titer in
the serum of various animals immunized by SpA Immunization by
vaccine group Control group F(ab').sub.2 F(ab').sub.2 fragment
Common fragment Common Animal secondary secondary Animal secondary
secondary species No. antibody antibody species No. antibody
antibody BALB/C 1 1:64000 1:128000 BALB/C 1 -- 1:128000 mouse mouse
BALB/C 2 1:128000 1:128000 BALB/C 2 -- 1:64000 mouse mouse BALB/C 3
1:128000 1:256000 BALB/C 3 -- 1:128000 mouse mouse BALB/C 4 1:64000
1:128000 BALB/C 4 -- 1:128000 mouse mouse BALB/C 5 1:128000
1:256000 BALB/C 5 -- 1:64000 mouse mouse BALB/C 6 1:128000 1:256000
BALB/C 6 -- 1:128000 mouse mouse BALB/C 7 1:256000 1:256000 BALB/C
7 -- 1:128000 mouse mouse BALB/C 8 1:128000 1:128000 BALB/C 8 --
1:128000 mouse mouse BALB/C 9 1:128000 1:256000 BALB/C 9 --
1:128000 mouse mouse BALB/C 10 1:64000 1:128000 BALB/C 10 --
1:64000 mouse mouse SD rat 1 1:128000 1:256000 SD rat 1 -- 1:128000
SD rat 2 1:64000 1:128000 SD rat 2 -- 1:128000 SD rat 3 1:128000
1:128000 SD rat 3 -- 1:128000 SD rat 4 1:128000 1:256000 SD rat 4
-- 1:128000 SD rat 5 1:32000 1:128000 SD rat 5 -- 1:64000 big ear
white 1 1:256000 1:256000 big ear white 1 -- 1:128000 rabbit rabbit
big ear white 2 1:256000 1:256000 big ear white 2 -- 1:256000
rabbit rabbit
Based on the Examples above, related detection kits can be easily
prepared by the one skilled in the art by using the detection
method provided herein according to the knowledge in the field, for
evaluation of the specific antibody level after immunized by the
SpA5 mutant protein and for assessment of the immunoprotective
effects.
C. Vaccine Formulations and the Preparation Methods Thereof
The vaccine formulations of the invention were based on the SpA5
proteins of the invention, and HI, MntC and mSEB proteins.
In one embodiment, the vaccine formulations of the invention
includes the SpA5 proteins; and in another embodiment, the vaccine
formulations of the invention includes the SpA5 proteins, and one
or more of MntC, mSEB and HI proteins.
Preferably, the sequence of the SpA5 protein was selected from any
one of SEQ ID NO. 1-4; the sequence of MntC protein was shown in
SEQ ID NO. 13; the sequence of mSEB protein was shown in SEQ ID NO.
14; and the sequence of HI protein was shown in SEQ ID NO. 15. More
preferably, in the vaccine formulations of the invention, the SpA5
protein was SpA5 (KKAA) or SpA5 (RRVV).
In a preferable embodiment, the antigen in the vaccine formulations
of the invention consists of SpA5, MntC, mSEB and HI proteins. The
concentration of each antigen was in the range from 10-100
.mu.g/ml, and preferably, was 50 .mu.g/ml.
Preferably, the vaccine formulations further comprises an adjuvant,
and preferably, the adjuvant was an aluminium adjuvant, such as
aluminum phosphate or aluminium hydroxide. The reagents used to
prepare the vaccine formulations could be easily determined by the
one skilled in the art.
The vaccine formulations above contains a plurality of antigens
screened from 2742 open reading frames (ORFs) in the whole genome
of Staphylococcus aureus by the inventors through reverse
vaccinology, high-throughput immunodominant antigenome, highly
efficient expression and purification of soluble protein and
through a large number of experiments for evaluating the
immunoprotective effects on animals, wherein the screened antigens
has strong antigenicity, favorable specificity and conservation,
and good protective effects. The antigens includes:
.alpha.-hemolysin (Hla), iron-regulated surface determinant protein
B (IsdB), Staphylococcus aureus protein A (SpA), enterotoxin B
(SEB), and manganese ion-binding protein C (MntC). These antigens
plays an important role in key points of Staphylococcus aureus
infection and immunologic escape. In the present invention, the
recombinant Staphylococcus aureus vaccines containing the antigens
above and the compositions thereof were successfully prepared,
based on structure analysis, molecular fusion, selection of
multiple components, and optimization components compatibility.
Invasion by Staphylococcus aureus infections can be effectively
protected against by the vaccines, since they blocked the metabolic
pathway of Staphylococcus aureus, inhibited adhesion and
colonization, controlled the diffusion of toxins, and destroyed
immunologic escape.
Due to complex antigen components in the vaccines of the invention,
and varied properties of each antigen, it was difficult to prepare
a single component vaccine formulation directly using one antigen.
The method for preparing a single component vaccine formulation was
complicated, with undesired immune effect, so it was not suitable
for industrial production. Besides, the adsorption rate by the
aluminium adjuvant varied among different antigens, and the
homogeneity in adsorption was also different, so that higher dosage
was usually desired for vaccination, while the immunoprotective
effects were often not desired. Additionally, the physicochemical
properties of the aluminium adjuvant might change during
preparation, resulting in ineffective immunization by the vaccine
formulations.
Principal problems includes: (1) no unified evaluation criteria for
the mixing ratio between the antigen and the element aluminium in
the prior art, in which extremely high content of element aluminium
was used to obtain a higher adsorption rate, which was harmful to
the health of patients, went against related requirements of WHO
and in "Guidelines for Preclinical Research on Preventive
Vaccines", and furthermore, did not conform to the trend in
pharmaceutical industry. (2) The existing procedure for vaccine
formulation was in an order of diluting the antigens and the
adjuvants separately, adsorbing, mixing and subpackaging, which was
feasible for formulating a single antigen component, but not for
multi-component formulations, due to enhanced difficulties and
costs with increased number of antigen components. (3) To address
the problems above, a method, in which the diluted adjuvant was
directly added to the antigen, was proposed, however, the
adsorption homogeneity could not be guaranteed for multi-component
formulation by this method. (4) There was no scientific and
rigorous evaluation system in the field of formulation; in the
prior art, only the preparation method for the adjuvant was
focused, while the formulation process was often neglected; for the
evaluation indices, attentions were usually paid to the adsorption
rate, while other indices were generally ignored, such as the
adsorption mode, the adsorption efficiency, the adsorption
homogeneity and the detection methods thereof, and the stability of
various parameters in the formulation process etc. (5) For the
histidine solution at present, the applicability was still
restricted for the proteins of different properties, although
parameters, such as histidine concentration and pH value etc., were
investigated; while in some vaccine formulation processes,
solubilizing agents, such as Tween, were added, it had already been
less used in the FDA approved medicines due to problems such as
potential safety hazard, and unfortunately no novel solubilizing
agent was reported yet. (6) In conventional formulation processes,
horizontal mixing mode was usually used for adsorption, and
innovation and development in techniques and equipments were still
demanded.
Accordingly, there was a demand to improve and innovate the process
related to the vaccine formulation processes at present, so that
key bottlenecks in the process of vaccine formulation could be
efficiently settled, such as the adsorption rate of antigen
protein, the adsorption homogeneity of multiple antigen components,
the stability, the reasonable and effective adsorption mode, making
the vaccine formulation process (especially for multi-component
vaccine formulation) simpler, more stable and more efficient, with
reduced production costs.
Thus, in another aspect, the present invention provided a method
for preparing a multi-component vaccine, which comprises diluting
the aluminium adjuvant and the vaccine antigen protein separately,
followed by mixing and/or adsorbing, specifically includes:
Method 1: individual vaccine antigen proteins were diluted by the
vaccine diluent separately, and then each mixed with the aluminum
phosphate adjuvant of an equal volume diluted by the vaccine
diluent, and adsorbed. Subsequently, the protein solutions were
mixed homogenously and subpackaged.
(1) According to the concentration of element aluminium (5.3 mg/ml)
in the aluminum phosphate adjuvant solution and the final
concentration of element aluminium (0.71 mg/ml) in the finished
vaccine product, the required volume of aluminum phosphate adjuvant
solution was calculated, exactly taken out, and added to a
formulation bottle. Vaccine diluent was then added to 50% of the
final volume of the vaccine formulation. The mixture was
sufficiently mixed.
(2) The final concentration of the recombinant Staphylococcus
aureus vaccine antigen proteins was 0.2 mg/ml, and the final
concentration of an individual recombinant Staphylococcus aureus
vaccine antigen protein was 0.05 mg/ml. According to the volume
required, the amount of each protein was calculated. Exact volume
of the individual recombinant Staphylococcus aureus vaccine antigen
protein solution was then added to a formulation bottle. Vaccine
diluent was then added to 12.5% of the final volume of the vaccine
formulation. The mixture was sufficiently mixed.
(3) Each diluted individual antigen protein solution and the
diluted adjuvant solution of the same volume were added to
subpackage bottles. The mixture was suspended vertically at 14 rpm
for adsorption for 1 h at ambient temperature controlled in the
range from 4-37.degree. C.
(4) After adsorption, the formulation was prepared by fully mixing
4 antigen protein solutions.
Method 2: individual vaccine antigen proteins were diluted by the
vaccine diluent separately, and then each mixed with the diluted
aluminum phosphate adjuvant of an equal volume. Subsequently, the
protein solutions were mixed and adsorbed all together, and then
subpackaged.
(1) According to the concentration of element aluminium (5.3 mg/ml)
in the aluminum phosphate adjuvant solution and the final
concentration of element aluminium (0.71 mg/ml) in the finished
vaccine product, the required volume of aluminum phosphate adjuvant
solution was calculated, exactly taken out, and added to a
formulation bottle. Vaccine diluent was then added to 50% of the
final volume of the vaccine formulation. The mixture was
sufficiently mixed.
(2) The final concentration of the recombinant Staphylococcus
aureus vaccine antigen proteins was 0.2 mg/ml, and the final
concentration of an individual recombinant Staphylococcus aureus
vaccine antigen protein was 0.05 mg/ml. According to the volume
required, the amount of each protein was calculated. Exact volumes
of individual recombinant Staphylococcus aureus vaccine antigen
protein solutions were then added to formulation bottles. Vaccine
diluent was then added to 12.5% of the final volume of the vaccine
formulation. The mixture was sufficiently mixed.
(3) Each diluted individual antigen protein solution and the
diluted adjuvant solution of the same volume were added to
subpackage bottles. Subsequently, 4 antigen protein solutions were
mixed, and suspended vertically at 14 rpm for adsorption for 1 h at
ambient temperature controlled in the range from 4-37.degree.
C.
Method 3: multiple vaccine antigen proteins were mixed and diluted
by the vaccine diluent. After fully mixed, the solution was then
mixed with the diluted aluminum phosphate adjuvant solution of an
equal volume. After adsorption, the solution was subpackaged.
(1) According to the concentration of element aluminium (5.3 mg/ml)
in the aluminum phosphate adjuvant solution and the final
concentration of element aluminium (0.71 mg/ml) in the finished
vaccine product, the required volume of aluminum phosphate adjuvant
solution was calculated, exactly taken out, and added to a
formulation bottle. Vaccine diluent was then added to 50% of the
final volume of the vaccine formulation. The mixture was
sufficiently mixed.
(2) The final concentration of the recombinant Staphylococcus
aureus vaccine antigen proteins was 0.2 mg/ml, and the final
concentration of an individual recombinant Staphylococcus aureus
vaccine antigen protein was 0.05 mg/ml. According to the volume
required, the amount of each protein was calculated. Exact volumes
of 4 required recombinant Staphylococcus aureus vaccine antigen
protein solutions were then added to a formulation bottle. Vaccine
diluent was then added to 50% of the final volume of the vaccine
formulation. The mixture was sufficiently mixed.
(3) The above diluted antigen protein solution and the diluted
adjuvant solution were added to a subpackge bottle. The mixture was
suspended vertically at 14 rpm for adsorption for 1 h at ambient
temperature controlled in the range from 4-37.degree. C.
Preferably, the vertical suspension could be substituted by
horizontal suspension.
Preferably, the ratio between the diluted aluminum phosphate
adjuvant solution and the diluted antigen protein solution was
1:1.
Preferably, the aluminum phosphate adjuvant could be substituted by
aluminium hydroxide adjuvant.
Preferably, the mass ratio between the antigen protein and the
element aluminium was 1:1.98, at which the adsorption rate could
reach 90%-100%.
Preferably, the vaccine diluent used was histidine buffer, which
consists of histidine (preferably, at concentration of 10 mmol/L),
poloxamer 188 (preferably, 0.02%), and sodium chloride (preferably,
at concentration of 0.9%), at a preferable pH value of 6.0.
The vaccine formulation prepared according to the method of the
invention was stable for more than 8 weeks at the temperature
ranged from 4.degree. C. to 37.degree. C.
The advantages of the method provided herein includes:
(1) Based on the problems such as lack of or defective evaluation
system for formulation in the prior art, existing verification
indices have been optimized in the present invention, besides the
conventional parameter adsorption rate, indices including the
weight ratio of components, the adsorption efficiency, the
stability, the homogeneity and the detection methods thereof etc,
and the stability of the parameters mentioned, have been proposed;
and a set of evaluation system for formulation process was
established, mainly based on the indices including the adsorption
rate, the adsorption efficiency, the weight ratio of components,
the homogeneity, and the stability, so that the formulation process
could be scientifically, precisely and rapidly evaluated (note: the
adsorption efficiency refers to the minimum time used to achieve
the target indices, such as the adsorption rate, and the
homogeneity etc).
(2) By using the weight ratio of components, the adsorption
efficiency and amount of element aluminium could be exactly and
scientifically described; for an adsorption rate of 90%-100%, the
weight ratio between the antigen and the element aluminium was
1:1.98, far less than presently reported ratio of 1:45, suggesting
that the amount of element aluminium could be effectively reduced
for adsorbing the same amount of protein, which was beneficial to
the health of patients, and met the requirements of WHO and in
"Guidelines for Preclinical Research on Preventive Vaccines".
(3) Difficulties and costs could be effectively reduced for
multi-antigen component vaccine formulation by using the particular
formulation process of the invention.
(4) The homogeneity and stability of the formulation could be
guaranteed by using the particular formulation process of the
invention.
(5) For the vaccine diluent, various parameters for the histidine
buffer system were optimized in the present invention, and
preferably, poloxamer 188 was selected as the solubilizing agent,
which extended the applicability and reduced the risk of side
effect of vaccine diluent.
(6) For the existing formulation modes and equipments, vertical
suspension mode and corresponding equipment were originally
proposed for the adsorption in the present invention, and an
utility model patent was applied for the equipment (issued patent
No. 201220314436.4).
Based on the above analysis, the existing formulation process was
optimized and improved by a variety of improvements and
innovations, which reduced the process steps, decreased the costs
and improved the production efficiency. By investigating the
component processing step, improving the adsorption mode and
changing the composition of the vaccine diluent, the adsorption
rate of antigen protein and the weight ratio were improved, which
ensured the adsorption homogeneity of each component in the
vaccine, and prevented the physicochemical properties of the
aluminium adjuvant from changing during formulation, also reduced
the amount of element aluminium, improved production efficiency and
reduced costs. In this invention, the evaluation indices were
improved, and new indices and their detection methods were
proposed. Besides, a set of scientific and rigorous evaluation
system for formulation was established, filling the gap in this
field.
The antigen proteins and various reagents used in this section were
listed as follows:
1. Antigen Proteins
Recombinant Staphylococcus aureus vaccine antigen proteins were
supplied by Chongqing Yuanlun Biotechnology Co., Ltd, and Third
Military Medical University of Chinese People's Liberation Army.
The antigen proteins included SpA5 (KKAA) of the invention, and HI,
mSEB and MntC protein.
2. Reagents
The reagents and equipments, such as SDS-PAGE, and HPLC, etc., were
provided by Chongqing Yuanlun Biotechnology Co., Ltd.
The vaccine diluent: 10 mM histidine (Merck corporation, US,
pharmaceutical grade), 0.9% NaCl (Sichuan Kelun company,
physiological saline for injection) and 0.01% poloxamer 188 (Merck
corporation, US, pharmaceutical grade), pH 6.0, pyrogen-free.
PBS: potassium dihydrogen phosphate (KH.sub.2PO.sub.4) 0.2 g
(domestic reagent of analytical grade), disodium hydrogen phosphate
(Na.sub.2HPO.sub.4.12H.sub.2O) 2.9 g (domestic reagent of
analytical grade), sodium chloride (NaCl) 8.0 g (domestic reagent
of analytical grade), and potassium chloride (KCl) 0.2 g, adding
water to a final volume of 1000 mL, pH 7.4;
Aluminum phosphate adjuvant (concentration of element aluminium:
5.3 mg/ml) was an imported product with original packaging from
GENERAL CHEMICAL corporation, US.
3. Equipments
Large-scale vertically suspending instrument used for vaccine
formulation was of independent intellectual property rights, and
manufactured by Shanghai Geshi Corporation.
Example 16: Formulation of 4-Component Recombinant Staphylococcus
aureus Vaccine (1920 mL)
(1) 256 mL aluminum phosphate adjuvant was added to a formulation
bottle, to which 704 mL vaccine diluent was added to obtain a final
volume of 960 mL. The mixture was fully mixed.
(2) The final concentration of the recombinant Staphylococcus
aureus vaccine antigen protein was 0.2 mg/ml, and the final
concentration of an individual recombinant Staphylococcus aureus
vaccine antigen protein was 0.05 mg/ml. Accordingly, based on the
initial concentration of each antigen protein, the amount of each
protein was taken out. The volume required for HI protein was 96
mL, to which 144 mL vaccine diluent was added; the volume required
for SpA5 (KKAA) was 48 mL, to which 192 mL vaccine diluent was
added; the volume required for mSEB protein was 60 mL, to which 180
mL vaccine diluent was added; and the volume required for MntC
protein was 60 mL, to which 180 mL vaccine diluent was added. The
mixtures of each protein and corresponding diluent were added to
respective formulation bottles, and mixed sufficiently.
(3) Diluted individual antigen protein solutions were each added to
the diluted adjuvant solution of the same volume in individual
subpackage bottles; the mixture was suspended vertically at 14 rpm
for adsorption for 1 h at ambient temperature controlled in the
range from 4-37.degree. C.
(4) After adsorption, vaccine formulation was prepared by fully
mixing all 4 protein solutions.
Example 17: Formulation of 4-Component Recombinant Staphylococcus
aureus Vaccine (1200 mL)
(1) 160 mL aluminum phosphate adjuvant was added to a formulation
bottle, to which 440 mL vaccine diluent was added to obtain a final
volume of 600 mL. The mixture was fully mixed.
(2) The final concentration of the recombinant Staphylococcus
aureus vaccine antigen protein was 0.2 mg/ml, and the final
concentration of an individual recombinant Staphylococcus aureus
vaccine antigen protein was 0.05 mg/ml. Accordingly, based on the
required amount, corresponding protein solutions were taken out.
The volume required for HI protein was 60 mL, to which 90 mL
vaccine diluent was added; the volume required for SpA5 (KKAA) was
30 mL, to which 120 mL vaccine diluent was added; the volume
required for mSEB protein was 37.5 mL, to which 112.5 mL vaccine
diluent was added; and the volume required for MntC protein was
37.5 mL, to which 112.5 mL vaccine diluent was added. The mixtures
of each protein and corresponding diluent were added to respective
formulation bottles, and mixed sufficiently.
(3) Diluted individual antigen protein solutions were each added to
the diluted adjuvant solution of the same volume in individual
subpackage bottles. Subsequently, 4 antigen solutions were mixed,
and vertically suspended at 14 rpm for adsorption for 1 h at
ambient temperature controlled in the range from 4-37.degree.
C.
Example 18: Formulation of 4-Component Recombinant Staphylococcus
aureus Vaccine (600 mL)
(1) 80 mL aluminum phosphate adjuvant required was added to a
formulation bottle, to which 220 mL vaccine diluent was added to a
final volume of 300 mL. The mixture was fully mixed.
(2) The final concentration of the recombinant Staphylococcus
aureus vaccine antigen protein was 0.2 mg/ml, and the final
concentration of an individual recombinant Staphylococcus aureus
vaccine antigen protein was 0.05 mg/ml. Accordingly, based on the
initial concentration of each protein, the amount of each protein
required was calculated. The volume required was 30 mL for HI
protein, 15 mL for SpA5 (KKAA), 18.75 mL for mSEB protein, and
18.75 mL for MntC protein. All 4 proteins were added to a
formulation bottle, to which 217.5 mL vaccine diluent was added to
a final volume of 300 mL. The mixture was mixed sufficiently.
(3) The diluted antigen protein solution and the diluted adjuvant
solution of the same volume were added to a subpackage bottle. The
mixture was suspended vertically at 14 rpm for adsorption for 1 h
at ambient temperature controlled in the range from 4-37.degree.
C.
Example 19: Adsorption Homogeneity and Completeness of the Antigen
Proteins in the Recombinant Staphylococcus aureus Vaccine by the
Aluminum Phosphate Adjuvant Characterized by SDS-PAGE
(1) 1 mL sample was each collected from the vaccine formulations of
Example 16-18, and centrifugated at 6000 rpm for 5 min at 4.degree.
C. The supernatant was carefully withdrawn, from which 40 .mu.l
sample was collected.
(2) The dissociation solution (1 M Na.sub.2CO.sub.3) of the same
volume as the supernatant was added to the precipitate. The
precipitate was re-suspended, and vertically suspended at room
temperature for 1 h. 40 .mu.l sample was collected.
(3) According to the formulation method of Example 18, a protein
solution free of the aluminum phosphate adjuvant was prepared, in
which the volume for aluminum phosphate was supplemented by the
vaccine diluent. After fully mixed, 40 .mu.l sample was
collected;
(4) To the sample collected, 10 .mu.l 5.times. loading buffer was
added. The mixture was heated at 100.degree. C. for 5 min. After
cooling down, the samples were centrifugated instantly, and 10
.mu.l samples were loaded to the gel.
(5) The procedure for 12% SDS-PAGE included initially running at 80
v for 20 min, and then at 180 v for 40 min. After running, the gel
was stained in Coomassie staining solution under shaking, followed
by destained in destaining solution under shaking. The gel was
observed in an imaging system. The results were shown in FIG. 43,
which indicated that the proteins could be fully adsorbed by the
aluminum phosphate adjuvant in all 3 methods, and no significant
difference was observed for adsorption. However, both methods 2 and
3 were simpler than method 1. If method 1 was employed to prepare a
multi-antigen vaccine, the equipments for adsorption and mixing
were required for each antigen, and could not be shared, so that
the total number of equipments used was much more than that for
both methods 2 and 3.
Example 20: Studies on the Weight Ratio Between the Components of
the Recombinant Staphylococcus aureus Vaccine
(1) 720 .mu.l aluminum phosphate adjuvant solution (concentration
of element aluminium: 5.3 mg/ml) was added to a formulation bottle,
into which 6.48 mL vaccine diluent was added and fully mixed. The
mixture was divided into 12 aliquots (600 .mu.l for each aliquot,
containing 60 .mu.l aluminum phosphate and 318 .mu.g element
aluminium).
(2) Based on dose escalation of each antigen used, 3 groups,
including 80 .mu.g dose group, 120 .mu.g dose group, and 160 .mu.g
dose group, were divided for each antigen, and there were totally
12 groups for all 4 antigens. The volume required for each protein
was calculated according to its concentration, taken out and added
to corresponding bottles, to which the vaccine diluent was added to
a final volume of 600 .mu.l.
(3) The diluted individual antigen protein solutions were each
added to the diluted adjuvant solution of the same volume in
individual subpackage bottles. The mixture was suspended vertically
at 14 rpm for adsorption for 1 h at ambient temperature controlled
in the range from 4-37.degree. C.
Example 21: The Weight Ratio Between the Components of the
Recombinant Staphylococcus aureus Vaccine Characterized by
SDS-PAGE
(1) 12 samples from Example 20 were centrifugated at 6000 rpm for 5
min at 4.degree. C. The supernatant was carefully withdrawn, from
which 40 .mu.l sample was collected.
(2) According to the formulation method of Example 20, a protein
solution free of the aluminum phosphate adjuvant was prepared, in
which the volume for aluminum phosphate was supplemented by the
vaccine diluent. After fully mixed, 40 .mu.l sample was
collected.
(3) To the sample collected, 10 .mu.l 5.times. loading buffer was
added. The mixture was heated at 100.degree. C. for 5 min. After
cooling down, the samples were centrifugated instantly, and 10
.mu.l samples were loaded to the gel.
(4) The procedure for 12% SD S-PAGE included initially running at
80 v for 20 min, and then at 180 v for 40 min. After running, the
gel was stained in Coomassie staining solution under shaking,
followed by destained in destaining solution under shaking. The gel
was observed in an imaging system. The results were shown in FIGS.
44 and 45, which indicated that for HI, SpA5 (KKAA) and MntC, 100%
adsorption of 160 .mu.g protein could be achieved by 318 .mu.g
element aluminium, i.e., the weight ratio between the protein and
element aluminium was 1:1.9875 for 100% adsorption; while for mSEB,
100% adsorption of 80 .mu.g protein and 90% adsorption of 160 .mu.g
protein could be achieved by 318 .mu.g element aluminium, i.e., the
weight ratio between the protein and element aluminium was 1:3.975
for 100% adsorption, and 1:1.9875 for 90% adsorption. Accordingly,
it was believed that for the recombinant Staphylococcus aureus
vaccine antigen protein, 90-100% adsorption could be achieved at
the weight ratio of 1:1.9875 between the protein and element
aluminium.
Meanwhile, it should also be pointed out that if only the volume
was described while the weight was neglected, i.e., the ratio was
not defined, the ratio between the protein and element aluminium
would be much lower, even lower than 1:1.
Example 22: Homogeneity of the Recombinant Staphylococcus aureus
Vaccine Characterized by SDS-PAGE
(1) 6 samples were randomly collected from the solution in Example
18 after complete adsorption and mixing well. The sampling location
and time were completely random. For each sample, 1 mL was
collected.
(2) The sample was centrifugated at 6000 rpm for 5 min at 4.degree.
C. The supernatant was carefully withdrawn, from which 40 .mu.l
sample was collected.
(3) The dissociation solution of the same volume as the supernatant
was added to the precipitate. The precipitate was re-suspended, and
vertically suspended at room temperature for 1 h. 40 .mu.l sample
was collected.
(4) To the sample collected, 10 .mu.l 5.times. loading buffer was
added. The mixture was heated at 100.degree. C. for 5 min. After
cooling down, the samples were centrifugated instantly, and 10
.mu.l samples were loaded to the gel.
(5) The procedure for 12% SDS-PAGE included initially running at 80
v for 20 min, and then at 180 v for 40 min. After running, the gel
was stained in Coomassie staining solution under shaking, followed
by destained in destaining solution under shaking. The gel was
observed in an imaging system. The results were shown in FIGS. 46
and 47, which indicated that no band was seen for the supernatant
of the sample after centrifugation, whereas the positions,
grayscale values and areas of the bands were all identical for the
precipitate after dissociation, suggesting that the amounts and
types of antigen proteins adsorbed by the adjuvant at each random
sampling point were totally the same, i.e., the homogeneity was
obtained for the adsorption in formulation process.
Example 23: Adsorption Rate and Efficiency for the Recombinant
Staphylococcus aureus Vaccine Antigen Protein Formulation
(1) 1.2 mL aluminum phosphate adjuvant solution (concentration of
element aluminium: 5.3 mg/ml) was added to a formulation bottle,
into which 10.8 mL vaccine diluent was added and fully mixed. The
mixture was divided into 20 aliquots (600 .mu.l for each aliquot,
containing 60 .mu.l aluminum phosphate and 318 .mu.g element
aluminium).
(2) There were 20 groups divided, including 0.5 h, 1 h, 2 h, 4 h,
and 8 h group for each antigen, based on the doubled adsorption
durations for each antigen protein. According to the
concentrations, the required volumes were calculated for each
protein, and added to corresponding bottles, into which the vaccine
diluent was added to a final volume of 600 .mu.l.
(3) The diluted individual antigen protein solutions were each
added to the diluted adjuvant solution of the same volume in
individual subpackage bottles. The mixture was suspended vertically
at 14 rpm for adsorption for a specified duration. The samples were
treated after adsorption at ambient temperature controlled in the
range from 4-37.degree. C.
Example 24: Adsorption Rate and Efficiency for the Recombinant
Staphylococcus aureus Vaccine Formulation Characterized by
SDS-PAG
(1) 20 samples of Example 23 were centrifugated at 6000 rpm for 5
min at 4.degree. C. The supernatant was carefully withdrawn, from
which 40 .mu.l sample was collected.
(2) To the sample collected, 10 .mu.l 5.times. loading buffer was
added. The mixture was heated at 100.degree. C. for 5 min. After
cooling down, the samples were centrifugated instantly, and 10
.mu.l samples were loaded to the gel.
(3) The procedure for 12% SDS-PAGE included initially running at 80
v for 20 min, and then at 180 v for 40 min. After running, the gel
was stained in Coomassie staining solution under shaking, followed
by destained in destaining solution under shaking. The gel was
observed in an imaging system. The results were shown in FIGS. 48
and 49, which indicated that for the recombinant Staphylococcus
aureus vaccine antigen protein, 100% adsorption could be achieved
within 2 h, and more than 90% adsorption within 1 h, i.e., 90-100%
adsorption of the recombinant Staphylococcus aureus vaccine antigen
protein could be achieved within 1 h.
Example 25: Stability of the Recombinant Staphylococcus aureus
Vaccine Characterized by SDS-PAGE
(1) The solution of Example 18 after adsorption and mixing well was
divided into 1 mL aliquots, and then encapsulated into 2 mL aseptic
penicillin vials. The vial was subsequently sealed by a cap and
stored at 37.degree. C.
(2) 3 vials were randomly taken out every 4 weeks. The samples were
centrifugated at 6000 rpm for 5 min at 4.degree. C. The supernatant
was carefully withdrawn, from which 40 .mu.l sample was
collected.
(3) The dissociation solution of the same volume as the supernatant
was added to the precipitate. The precipitate was re-suspended, and
vertically suspended at room temperature for 1 h. 40 .mu.l sample
was collected.
(4) To the sample collected, 10 .mu.l 5.times. loading buffer was
added. The mixture was heated at 100.degree. C. for 5 min. After
cooling down, the samples were centrifugated instantly, and 10
.mu.l samples were loaded to the gel.
(5) The procedure for 12% SDS-PAGE included initially running at 80
v for 20 min, and then at 180 v for 40 min. After running, the gel
was stained in Coomassie staining solution under shaking, followed
by destained in destaining solution under shaking. The gel was
observed in an imaging system. The results were shown in FIGS. 50,
51 and 52, which indicated that no band was seen for the
supernatant of the sample after centrifugation, whereas the
positions, grayscale values and areas of the bands were all
identical for the precipitate after dissociation, suggesting that
the amounts and types of antigen proteins adsorbed by the adjuvant
at each random sampling point were totally the same. The results
further confirmed the homogeneity of the formulation. Besides, it
also demonstrated the stability of the formulation, including the
stability of the adsorption rate, the homogeneity and the antigen
protein.
As demonstrated by the results, the formulation of the invention
was stable for at least 12 weeks under accelerated temperature
condition (37.degree. C.), which further confirmed the
effectiveness and stability of the formulation process.
Example 26: Immunization Potency and Protection Effect Against
Challenging for the Vaccines of Various Components
Multi-component vaccines were prepared using SpA5 (KKAA) as the
antigen candidate, in monovalent, bivalent, trivalent and
tetravalent combination with HI, MntC, and mSEB. The antibody titer
was evaluated after further immunization of mice. ELISA was
performed for the serum, and the results were listed in Table
10.
Protection against challenging was tested (the processes for
immunization and protection against challenging were as described
in Example 12). 6 rounds of experiments were carried out from V34
to V39, in which there were 20 Balb/C mice (6-week old, Beijing HFK
Bioscience Co., Ltd.) in each group. Immunization was performed by
intramuscular injection, with a protein content of 30 .mu.g for
each component. The procedure of challenging test was as follows:
immunization was performed by quadriceps femoris injection (Day 0,
14 and 21) of each vaccine for 3 times; after the last
immunization, viable Staphylococcus aureus MRSA-252 was injected
via caudal vein at a fatal dose on Day 14 for the challenging test.
The amount of bacteria suspension was 1.25.times.10.sup.9 CFU for
each BALB/C mouse. After observed for 10 days, the survival rate
was calculated for each group. The results were listed in table
11.
TABLE-US-00010 TABLE 10 The antibody titers in the mice after
immunization by various proteins and their combinations as detected
by ELISA Antibody titer .times. 10.sup.5 Groups V34 V35 V36 V37 V38
V39 Average Control Vaccine diluent 0.001 0.001 0.001 0.001 0.001
0.001 0.001 AlPO.sub.4 0.001 0.001 0.001 0.001 0.001 0.001 0.001
Monovalent HI 1.28 1.28 1.11 1.28 1.19 1.28 1.24 mSEB 1.11 1.28
1.04 1.19 1.04 1.11 1.13 SpA5 0.84 0.79 0.74 0.84 0.69 0.79 0.78
MntC 1.28 1.28 1.28 1.28 1.28 1.28 1.28 Bivalent HI + mSEB HI 1.11
1.04 0.97 0.91 0.97 1.19 1.03 mSEB 1.19 1.11 1.04 1.19 0.97 1.04
1.09 HI + SpA5 HI 1.11 1.04 1.11 0.91 0.97 1.11 1.04 SpA5 0.49 0.45
0.52 0.49 0.60 0.56 0.52 HI + MntC HI 1.11 1.19 1.11 0.92 1.04 0.97
1.06 MntC 1.28 1.28 1.28 1.28 1.28 1.28 1.28 mSEB + SpA5 mSEB 0.91
1.19 1.04 0.97 1.19 1.11 1.07 SpA5 0.52 0.56 0.64 0.52 0.60 0.56
0.57 mSEB + MntC mSEB 1.19 1.10 1.02 0.94 1.10 1.02 1.06 MntC 1.28
1.28 1.28 1.28 1.28 1.28 1.28 SpA5 + MntC SpA5 0.49 0.52 0.56 0.49
0.64 0.60 0.55 MntC 1.28 1.28 1.28 1.28 1.28 1.28 1.28 Trivalent HI
+ mSEB + SpA5 HI 0.97 1.11 0.91 1.04 1.19 0.97 1.03 mSEB 1.04 0.97
1.04 0.91 0.84 1.04 0.97 SpA5 0.55 0.56 0.52 0.52 0.49 0.52 0.53 HI
+ mSEB + HI 1.11 0.91 0.91 1.11 0.97 1.04 1.01 MntC mSEB 0.85 0.8
0.91 0.74 0.69 0.74 0.79 MntC 1.28 1.28 1.28 1.28 1.28 1.28 1.28 HI
+ SpA5 + HI 0.69 0.79 0.64 0.69 0.60 0.74 0.69 MntC SpA5 0.24 0.26
0.30 0.26 0.28 0.24 0.26 MntC 1.28 1.28 1.28 1.28 1.28 1.28 1.28
mSEB + SpA5 + mSEB 0.91 0.84 0.79 1.04 0.84 0.79 0.87 MntC SpA5
0.24 0.23 0.24 0.20 0.21 0.23 0.23 MntC 1.28 1.28 1.28 1.28 1.28
1.28 1.28 Tetravalent HI + SpA5 + HI 0.79 0.74 0.79 0.84 0.79 0.69
0.77 mSEB + MntC Spa5 0.23 0.20 0.23 0.21 0.21 0.20 0.21 mSEB 0.37
0.34 0.37 0.34 0.37 0.32 0.35 MntC 1.28 1.28 1.28 1.28 1.28 1.28
1.28
TABLE-US-00011 TABLE 11 Evaluation on the protection effects
against challenging in the mice after immunization by various
vaccines Survival rate (%) Groups V34 V35 V36 V37 V38 V39 Average
Control Vaccine diluent 0% 10% 10% 20% 10% 30% 13.3% AlPO.sub.4 30%
40% 50% 30% 30% 30% 35% Monovalent HI 30% 30% 40% 30% 30% 30% 31.7%
mSEB 30% 11.1% 30% 30% 30% 30% 26.9% SpA5 70% 20% 30% 30% 40% 30%
36.7% MntC 60% 10% 30% 30% 40% 30% 33.3% Bivalence HI + mSEB 50%
37.5% 30% 60% 80% 90% 57.9% HI + SpA5 50% 40% 50% 60% 80% 80% 60%
HI + MntC 60% 50% 25% 80% 90% 100% 67.5% mSEB + SpA5 70% 90% 60%
80% 100% 60% 76.7% mSEB + MntC 70% 70% 70% 70% 100% 90% 78.3% SpA5
+ MntC 70% 70% 80% 70% 60% 90% 73.3% Trivalent HI + mSEB + SpA5 70%
100% 77.8% 70% 80% 80% 79.6% HI + mSEB + MntC 70% 60% 50% 80% 70%
100% 71.7% HI + SpA5 + MntC 70% 80% 88.9% 70% 60% 60% 71.5% mSEB +
SpA5 + MntC 80% 70% 90% 80% 70% 90% 80% Tetravalent HI + SpA5 +
mSEB + MntC 90% 80% 90% 90% 90% 90% 88.3%
It was indicated by the results that the best protection effect was
observed for SpA5 (KKAA) in the monovalent groups; and in the
bivalent groups, the survival rates were greatly enhanced in the
groups containing SpA5; while in the trivalent groups containing
SpA5 (mSEB+SpA5+MntC), the protection effect was further enhanced;
and the immunoprotective effect on the challenged animals of the
tetravalent group containing SpA5 was the best among all
groups.
Example 27: Optimization of the Animal Immunization Procedure
In order to make the recombinant Staphylococcus aureus vaccine more
suitable for clinical practice, the immunization procedure was
optimized for the finally determined vaccine components
HI+MntC+mSEB+SpA5 (30 .mu.g for each component) as listed in the
table below.
The animals were used as described in Example 12. For each vaccine,
the groups for the immunization procedure were divided into the
diluted vaccine protein groups and the experiment groups, with 30
animals in each group. The immunization procedure was listed in
Table 12.
TABLE-US-00012 TABLE 12 Immunization procedure Times for
immunization 3 2 1 Immunization procedure (Day) 0, 3, 7 0, 7 0 0,
3
Protection effect against challenging was listed in Table 13.
TABLE-US-00013 TABLE 13 Protection effect against challenging on
animals using various immunization procedures Number of survival
animals after Immunization 10 days Survival rate procedure Vaccine
diluent Vaccine Vaccine diluent Vaccine 0, 3, 7 0 29 0 97% 0, 7 0
27 0 90% 0, 3 0 26 0 87% 0 0 21 0 70%
As indicated by the results, the best protection effect on the
animals after challenging was obtained by the immunization
procedure on Day 0, 3 and 7, which was also suitable for clinical
application. By this experiment, the effectiveness of the
4-component recombinant Staphylococcus aureus vaccine was further
confirmed.
Example 28: Acute Toxicity Test on Mice
The test was performed by JOINN Laboratories (Suzhou), Inc.,
similarly hereinafter.
1) Test Samples, Adjuvant Control and Negative Control
(1) Test Samples
Name: recombinant Staphylococcus aureus vaccine
Concentration/content: total content of antigens: 120 .mu.g/0.6 mL,
containing 30 .mu.g/0.6 mL HI; 30 .mu.g/0.6 mL SpA5; 30 .mu.g/0.6
mL MntC; and 30 .mu.g/0.6 mL mSEB.
(2) Adjuvant Control
Name: adjuvant control for the recombinant Staphylococcus aureus
vaccine
Supplier: Chongqing Yuanlun Biotechnology Co., Ltd.
Lot Number: 20130516
Content of aluminium: 0.72 mg/ml.
(3) Negative Control Name: Sodium Chloride Injection
Manufacturer: Jiangsu Yabang Shengyuan Pharmaceutical Co., Ltd.
Lot Number: 12120104.
2) Experiment Animals: ICR Mice (SPF Grade, JOINN Laboratories
(Suzhou), Inc.)
Number: 40 mice (20 mice in each group, with half male and half
female mice)
Administration frequency: single administration
Route of administration: intramuscular injection
Dosage: 10 mL/kg
TABLE-US-00014 TABLE 14 Group division and administration scheme
for the acute toxicity test Number of Dosage a Volume b the animal
Groups (dose/mouse) (mL/mouse) Male Female 1 Negative control 0 0.6
10 10 group 2 Test sample 1 0.6 10 10 group Note: the total content
of antigen administrated in the test sample groups was 6000
.mu.g/kg, which was 3000 folds of the intended dosage for clinical
practice.
3) Test Results
During test, no dead or dying animal was observed in each group. No
abnormal reaction was observed in clinical in the animals of each
group. As compared with the animals of the same gender at the same
time in the negative control group, no administration-related
toxicologically regular change was observed for the body weight and
the food intake in the animals in each group. The results were
shown in FIGS. 53 and 54.
Pathological examination: after euthanasia, no significant
administration-related abnormal change was observed for all animals
during general observation; gross anatomy was performed to all
animals, in which swelling was seen in the uterus (bilateral) of
only 2 female mice in the negative control group, which was just
normal physiologically periodic change.
Example 29: Systemic Active Anaphylaxis of Guinea Pig
1) The test samples, adjuvant control and negative control were the
same as described in Example 28.
Positive control
Name: human serum albumin
Lot Number: 201206043
Manufacturer: Shanghai Institute of Biological Products Co.,
Ltd.
Reason for selection: the product was a known positive sensitizer
for guinea pigs
2) Experiment Methods
Animals: Hartley guinea pigs (SPF grade, Beijing Charles River
Laboratories. Inc.)
Quantity: 24 guinea pigs (6 guinea pigs in each group, with half
male and half female mice)
Route of administration: intramuscular injection for sensitization,
and foot intravenous injection for stimulation.
TABLE-US-00015 TABLE 15 Group division and administration scheme
Groups Male Female Sensitization (i.m) Stimulation (i.v.) 1
Negative control 1-3 4-6 Once every other 14 days after the group
day for totally 3 last sensitization times 2 Positive control group
7-9 10-12 Once every other 14 days after the day for totally 3 last
sensitization times 3 Low dosage group of 13-15 16-18 Once every
other 14 days after the the test sample day for totally 3 last
sensitization times 4 High dosage group of 19-21 22-24 Once every
other 14 days after the the test sample day for totally 3 last
sensitization times
TABLE-US-00016 TABLE 16 Intended dosages Sensitization (i.m)a
Admin- istration Stimulation (i.v.)b Groups dosage Volume Dosage
Volume 1 Negative -- 0.6 mL/ -- 1.2 mL/ control group animal animal
2 Positive control 18 mg/ 0.6 mL/ 36 mg/ 1.2 mL/ group animal
animal animal animal 3 Test samples 0.1 dose/ 0.06 mL/ 0.2 dose/
0.12 mL/ Low dosage animal animal animal animal group 4 Test
samples 1 dose/ 0.6 mL/ 2 dose/ 1.2 mL/ High dosage animal animal
animal animal group Note: the dosages of the test samples for
sensitization were 30 .mu.g/kg and 300 .mu.g/kg, respectively, in
low and high dosage groups, corresponding to 15 and 150 folds of
the intended clinical dosage.
3) Results: during sensitization, no abnormal reaction was observed
in all animals.
TABLE-US-00017 TABLE 17 Summary of allergic reactions in animals in
each group after stimulation Number of the animals suffering
allergy at different levels Extremely Number of Weakly Strongly
strongly Groups the animals Negative positive Positive positive
positive Negative control 6 6 0 0 0 0 group Positive control 6 0 0
0 5 1 group Low dosage group 6 0 0 0 1 5 of the test sample High
dosage 6 0 0 0 0 6 group of the test sample
Under the test conditions, the recombinant Staphylococcus aureus
vaccine was intramuscularly injected for sensitization at a dosage
of 0.1 dose/animal (corresponding to 15 folds of the intended
clinical dosage) and 1 dose/animal (corresponding to 150 folds of
the intended clinical dosage) (the total content of proteins in 1
dose was 120 .mu.g), and intravenously injected at a dosage of 0.2
dose/animal and 2 dose/animal for stimulation, so that immediate
allergy could be resulted in guinea pigs. The results were
consistent with the features of allergy in the guinea pig caused by
a common purified protein vaccine (such as tetanus vaccine, and
diphtheria vaccine etc.).
Example 30: Toxicity Test of 4-Week Repeated Intramuscular
Injection to SD Rats and 4-Week Recovery
1) The test samples, adjuvant control and negative control were the
same as described in Example 28.
2) Experiment Methods
Animals: SD rats (SPF grade, Beijing Charles River Laboratories.
Inc.)
Route of administration: intramuscular injection
Administration frequency and period: administration once on D1,
D15, D22 and D29, for totally 4 times.
TABLE-US-00018 TABLE 18 Experiment scheme: Dosage a Volume b Number
of the No. Groups (dose/animal) (mL/animal) animals/gender c Male
Female Main test groups 1 Negative control group 0 1.8 10 + 5
13-2331~13-2345 13-2346~13-2360 2 Adjuvant control group 3 1.8 10 +
5 13-2361~13-2375 13-2376~13-2390 3 Low dosage group of the test
sample 0.3 0.18 10 + 5 13-2391~13-2405 13-2406~13-2420 4 Middle
dosage group of the test sample 1 0.6 10 + 5 13-2421~13-2435
13-2436~13-2450 5 High dosage group of the test sample 3 1.8 10 + 5
13-2451~13-2465 13-2466~13-2480 Satellite groups d 6 Negative
control group 0 1.8 5 13-2481~13-2485 13-2486~13-2490 7 Low dosage
group of the test sample 0.3 0.18 5 13-2491~13-2495 13-2496~13-2500
8 Middle dosage group of the test sample 1 0.6 5 13-2501~13-2505
13-2506~13-2510 9 High dosage group of the test sample 3 1.8 5
13-2511~13-2515 13-2516~13-2520 a. The intended clinical dosage was
0.6 mL/dose/time/person, and the dosage unit for rats was
"dose/animal". 1 dose corresponded to 1 clinical inoculation
dosage. b. The volume in the Table above was a theoretical
administration volume, and the actual administration volume was
kept in the original recording. c. The first 10
animals/gender/group were used for anatomy after administration for
4 weeks (D32), and the last 5 animals/gender/group were used for
anatomy after 4-week recovery(D57). d. The blood samples were
collected from the animals in the satellite groups only for
antibody and cytokine assays. Other data were stored in the source
materials, and not presented in the report.
3) Test Results
TABLE-US-00019 TABLE 19 Clinical observation (D1-D32) Dosage
umbilicus Gender Groups (dose/animal) n Sclerosis regionupheaval 1
0 15 0 0 2 3 15 9 0 3 0.3 15 3 0 4 1 15 14 1 5 3 15 15 0 1 0 15 0 0
2 3 15 4 0 3 0.3 15 1 0 4 1 15 4 0 5 3 15 12 0 Group 1: negative
control group; Group 2: adjuvant control group; Group 3: low dosage
group of the test sample; Group 4: middle dosage group of the test
sample; and Group 5: high dosage group of the test sample
TABLE-US-00020 TABLE 20 Clinical observation on the animas
(D32-D57) umbilicus Dosage region Gender Groups (dose/animal) n
Sclerosis upheaval 1 0 15 0 0 2 3 15 9 0 3 0.3 15 3 0 4 1 15 14 1 5
3 15 15 0 1 0 15 0 0 2 3 15 4 0 3 0.3 15 1 0 4 1 15 4 0 5 3 15 12 0
Group 1: negative control group; Group 2: adjuvant control group;
Group 3: low dosage group of the test sample; Group 4: middle
dosage group of the test sample; and Group 5: high dosage group of
the test sample
TABLE-US-00021 TABLE 21 Summary of the blood cell counts in the
male animals (3 days after the last administration, D32) Neut PLT
WBC Neutrophilic Dosage Thrombocyte Leucocyte granulocyte Groups
(dose/animal) (.times.10.sup.9/L) (.times.10.sup.9/L) (%) 1 0 Mean
.+-. SD 1301.4 .+-. 153.5 9.47 .+-. 2.17 11.58 .+-. 3.11 n 10 10 10
2 3 Mean .+-. SD 1441.6 .+-. 142.2 10.93 .+-. 1.69 12.48 .+-. 1.79
n 10 10 10 3 0.3 Mean .+-. SD 1301.9 .+-. 100.3 10.69 .+-. 2.20
13.57 .+-. 4.50 n 10 10 10 4 1 Mean .+-. SD 1401.7 .+-. 203.6 12.33
.+-. 2.54* 19.55 .+-. 3.01* n 10 10 10 5 3 Mean .+-. SD 1597.0 .+-.
219.6* 14.17 .+-. 3.33* 22.93 .+-. 4.64* n 10 10 10 Group 1:
negative control group; Group 2: adjuvant control group; Group 3:
low dosage group of the test sample; Group 4: middle dosage group
of the test sample; and Group 5: high dosage group of the test
sample
TABLE-US-00022 TABLE 22 Summary of the blood cell counts in the
female animals (3 days after the last administration, D32) Neut
Neutrophilic Dosage PLT WBC granulocyte Groups (dose/animal)
(.times.10.sup.9/L) (.times.10.sup.9/L) (%) 1 0 Mean .+-. SD 1379.0
.+-. 158.8 9.89 .+-. 4.84 9.16 .+-. 4.15 n 10 10 10 2 3 Mean .+-.
SD 1431.4 .+-. 143.5 11.77 .+-. 3.95 9.86 .+-. 2.38 n 10 10 10 3
0.3 Mean .+-. SD 1453.7 .+-. 129.0 9.82 .+-. 2.74 12.32 .+-. 4.74 n
10 10 10 4 1 Mean .+-. SD 1389.6 .+-. 163.6 12.38 .+-. 4.84 15.16
.+-. 3.28* n 10 10 10 5 3 Mean .+-. SD 1611.7 .+-. 154.3* 15.89
.+-. 4.17* 22.44 .+-. 6.77* n 10 10 10 Group 1: negative control
group; Group 2: adjuvant control group; Group 3: low dosage group
of the test sample; Group 4: middle dosage group of the test
sample; and Group 5: high dosage group of the test sample
As indicated by the body temperature, the T lymphocyte
subpopulation assay and the biochemical assay of blood, no
toxicologically abnormal change was observed in the animals of each
dosage group of the test sample, as compared with the negative
control group.
As compared with the negative control group, no test sample-related
toxicologically abnormal change was observed for the indices of the
male animals, including the organ weight, the organ/body weight
ratio and the organ/brain weight ratio.
Antibodies against each component in all dosage groups of the test
sample could be detected on Day 57.
TABLE-US-00023 TABLE 23 IgG antibody detection in the serum (HI)
Generation Testing Dosage rate of the time Groups (dose/animal) N
antibody D1 Negative control group 0 10 0/10 Low dosage group of
the test 0.3 10 0/10 sample Middle dosage group of the test 1 10
0/10 sample High dosage group of the test 3 10 0/10 sample D14
Negative control group 0 10 0/10 Low dosage group of the test 0.3
10 5/10 sample Middle dosage group of the test 1 10 2/10 sample
High dosage group of the test 3 10 6/10 sample D21 Negative control
group 0 10 0/10 Low dosage group of the test 0.3 10 7/10 sample
Middle dosage group of the test 1 10 10/10 sample High dosage group
of the test 3 10 9/10 sample D28 Negative control group 0 10 0/10
Low dosage group of the test 0.3 10 9/10 sample Middle dosage group
of the test 1 10 10/10 sample High dosage group of the test 3 10
10/10 sample D57 Negative control group 0 10 0/10 Low dosage group
of the test 0.3 10 10/10 sample Middle dosage group of the test 1
10 10/10 sample High dosage group of the test 3 10 10/10 sample
TABLE-US-00024 TABLE 24 IgG antibody detection in the serum (MntC)
Generation Testing Dosage rate of the time Groups (dose/animal) N
antibody D1 Negative control group 0 10 0/10 Low dosage group of
the test 0.3 10 0/10 sample Middle dosage group of the test 1 10
0/10 sample High dosage group of the test 3 10 0/10 sample D14
Negative control group 0 10 0/10 Low dosage group of the test 0.3
10 10/10 sample Middle dosage group of the test 1 10 10/10 sample
High dosage group of the test 3 10 10/10 sample D21 Negative
control group 0 10 0/10 Low dosage group of the test 0.3 10 10/10
sample Middle dosage group of the test 1 10 10/10 sample High
dosage group of the test 3 10 10/10 sample D28 Negative control
group 0 10 0/10 Low dosage group of the test 0.3 10 10/10 sample
Middle dosage group of the test 1 10 10/10 sample High dosage group
of the test 3 10 10/10 sample D57 Negative control group 0 10 0/10
Low dosage group of the test 0.3 10 10/10 sample Middle dosage
group of the test 1 10 10/10 sample High dosage group of the test 3
10 10/10 sample
TABLE-US-00025 TABLE 25 IgG antibody detection in the serum (mSEB)
Generation Testing Dosage rate of the time Groups (dose/animal) N
antibody D1 Negative control group 0 10 0/10 Low dosage group of
the test 0.3 10 0/10 sample Middle dosage group of the test 1 10
0/10 sample High dosage group of the test 3 10 0/10 sample D14
Negative control group 0 10 0/10 Low dosage group of the test 0.3
10 5/10 sample Middle dosage group of the test 1 10 9/10 sample
High dosage group of the test 3 10 9/10 sample D21 Negative control
group 0 10 0/10 Low dosage group of the test 0.3 10 10/10 sample
Middle dosage group of the test 1 10 10/10 sample High dosage group
of the test 3 10 10/10 sample D28 Negative control group 0 10 0/10
Low dosage group of the test 0.3 10 10/10 sample Middle dosage
group of the test 1 10 10/10 sample High dosage group of the test 3
10 10/10 sample D57 Negative control group 0 10 0/10 Low dosage
group of the test 0.3 10 10/10 sample Middle dosage group of the
test 1 10 10/10 sample High dosage group of the test 3 10 10/10
sample
TABLE-US-00026 TABLE 26 IgG antibody detection in the serum (SpA5
(KKAA) Generation Testing Dosage rate of the time Groups
(dose/animal) N antibody D1 Negative control group 0 10 0/10 Low
dosage group of the test 0.3 10 0/10 sample Middle dosage group of
the test 1 10 0/10 sample High dosage group of the test 3 10 0/10
sample D14 Negative control group 0 10 0/10 Low dosage group of the
test 0.3 10 10/10 sample Middle dosage group of the test 1 10 10/10
sample High dosage group of the test 3 10 10/10 sample D21 Negative
control group 0 10 0/10 Low dosage group of the test 0.3 10 10/10
sample Middle dosage group of the test 1 10 10/10 sample High
dosage group of the test 3 10 10/10 sample D28 Negative control
group 0 10 0/10 Low dosage group of the test 0.3 10 10/10 sample
Middle dosage group of the test 1 10 10/10 sample High dosage group
of the test 3 10 10/10 sample D57 Negative control group 0 10 0/10
Low dosage group of the test 0.3 10 10/10 sample Middle dosage
group of the test 1 10 10/10 sample High dosage group of the test 3
10 10/10 sample
Example 31: Toxicity Test of 4-Week Repeated Intramuscular
Injection to Cynomolgus Monkeys and 4-Week Recovery
1) The test samples, adjuvant control and negative control were the
same as described in Example 28.
2) Experiment Methods
Experiment animals: cynomolgus monkeys (ordinary grade, Hainan
experimental primate corporation)
Route of administration: intramuscular injection
Administration frequency and period: administration once on D1,
D15, D22 and D29, for totally 4 times.
TABLE-US-00027 TABLE 27 Experiment scheme Number of the Dosage a
Volume b animal c No. Groups (dose/animal) (mL/animal)
(animal/gender) Male Female 1 Negative control group 0 3 2 + 2
13-4161~13-4164 13-4165~13-4168 2 Adjuvant control group 5 3 2 + 2
13-4169~13-4172 13-4173~13-4176 3 Low dosage group of the test
sample 0.2 0.12 2 + 2 13-4177~13-4180 13-4181~13-4184 4 Middle
dosage group of the test sample 1 0.6 2 + 2 13-4185~13-4188
13-4189~13-4192 5 High dosage group of the test sample 5 3 2 + 2
13-4193~13-4196 13-4197~13-4200 a. The intended clinical dosage was
0.6 mL/dose/time/person, and the dosage unit for the monkeys was
"dose/animal". 1 dose corresponded to 1 clinical inoculation
dosage. b. The volume in the Table above was a theoretical
administration volume, and the actual administration volume was
kept in original recording. c. For each group, the first 2
animals/gender/group were used for anatomy on day 3 after the last
administration, and the last 2 animals/gender/group were used for
anatomy after 4-week recovery.
3) Test Results:
TABLE-US-00028 TABLE 28 Clinical observation (D1-D32) Loose Groups
Dosage (dose/animal) n stools Erythema Sclerosis 1 0 8 0 0 0 2 5 8
0 1 7 3 0.2 8 1 0 1 4 1 8 0 2 7 5 5 8 0 1 8
As indicated by the body weight, the body temperature, the T
lymphocyte subpopulation assay, the blood cell assay and the
biochemical assay of blood, no test sample-related toxicologically
abnormal change was observed, as compared with the negative control
group.
TABLE-US-00029 TABLE 29 Results of ELISPOT (D18) Dosage Groups
(dose/animal) HI MntC mSEB SpA5 1 0 Mean .+-. SD 33.8 .+-. 50.8
38.8 .+-. 69.8 36.3 .+-. 83.8 6.9 .+-. 19.4 n 8 8 8 8 2 5 Mean .+-.
SD 111.3 .+-. 247.1 195.6 .+-. 403.2 95.6 .+-. 250.5 6.9 .+-. 10.3
n 8 8 8 8 3 0.2 Mean .+-. SD 89.4 .+-. 135.3 142.5 .+-. 140.8 85.6
.+-. 151.6 28.1 .+-. 56.6 n 8 8 8 8 4 1 Mean .+-. SD 168.1 .+-.
312.3 459.4 .+-. 602.6 203.8 .+-. 272.3 290.6 .+-. 200.4* n 8 8 8 8
5 5 Mean .+-. SD 81.9 .+-. 121.2 29.4 .+-. 55.3 35.6 .+-. 81.3 35.6
.+-. 81.3 n 8 8 8 8 Group 1: negative control group; Group 2:
adjuvant control group; Group 3: low dosage group of the test
sample; Group 4: middle dosage group of the test sample; and Group
5: high dosage group of the test sample
Example 32: Toxicity Test on Reproductive Development of SD Rats
Administrated by Repeated Intramuscular Injection
1) The test samples, adjuvant control and negative control were the
same as described in Example 28.
2) Experiment Methods:
Animals: SD rats (SPF grade, Beijing Charles River Laboratories.
Inc.)
TABLE-US-00030 TABLE 30 Experiment scheme Number of the Dosage a
Volume b animals Groups (dose/animal) (mL/animal) Male Female c 1
Negative 0 1.8 28 28 + 28 control group 2 Adjuvant 3 1.8 28 28 + 28
control group 3 Low dosage 1 0.6 28 28 + 28 group of the test
sample 4 High dosage 3 1.8 28 28 + 28 group of the test sample
Note: the first 28 female animals in each group were used for
normal delivery until the stage of lactation is finished, and the
last 28 animals were used for anatomy on Day 20 of gestation (GD20)
to examine the fetal rats.
Administration frequency and period: the male rats were
administrated for 3 times (D1, D15 and D22) before mating; and the
female rats were also administrated for 3 times (D1, D15 and D22)
before mating. The male and female rats were raised in the same
cage for mating 1 week after administration of the last dose to the
male rats. The female rats were administrated once on Day 6 of
gestation (GD6).
3) Test Results
As compared with the negative control group, no toxicological
abnormal change was observed for the body weight of the animals in
each dosage groups of the test sample.
As compared with the negative control group, no toxicological
abnormal change was observed for the food intake of the animals in
each dosage groups of the test sample.
TABLE-US-00031 TABLE 31 Reproductive capacity of the parent male
and female rats High dosage Low dosage group of the Negative
control Adjuvant control group of the test test sample group group
sample (3 (0 dose/animal) (3 doses/animal) (1 dose/animal)
doses/animal) Number of the animals 28( ), 28 ( ) 28 ( ), 28 ( ) 28
( ), 28 ( ) 28 ( ), 28 ( ) in the same cage Mating rate of the male
100.0 (28/28) 100.0 (28/28) 100.0 (28/28) 100.0 (28/28) rats (%)
Fertility rate of the 85.7 (24/28) 78.6 (22/28) 85.7 (24/28) 78.6
(22/28) male rats (%) Mating rate of the 100.0 (28/28) 100.0
(28/28) 100.0 (28/28) 100.0 (28/28) female rats (%) Fertility rate
of the 85.7 (24/28) 78.6 (22/28) 85.7 (24/28) 78.6 (22/28) female
rats (%) Pregnancy rate (%) 85.7 (24/28) 78.6 (22/28) 85.7 (24/28)
78.6 (22/28) Gestation rate (%) 85.7 (24/28) 78.6 (22/28) 85.7
(24/28) 78.6 (22/28) Number of the female 5 5 2 7 rats of irregular
estrous cycle Rate of irregularly 17.9 (5/28) 17.9 (5/28) 7.1
(2/28) 25.0 (7/28) estrous rats (%) Days in the same cage 5.4 .+-.
4.9 4.5 .+-. 3.9 4.0 .+-. 3.1 4.8 .+-. 4.3 Days of mating 5.4 .+-.
4.9 4.5 .+-. 3.9 4.0 .+-. 3.1 4.8 .+-. 4.3
Detection Indices for the Parent Male Rats:
TABLE-US-00032 TABLE 32 Organ weight and coefficient of the parent
male rats Organ coefficient (%) Testicle Epididymis Testicle
Epididymis Groups (bilateral, g) (bilateral, g) Prostate (g)
(bilateral) (bilateral) Prostate Negative 3.123 .+-. 0.291 1.033
.+-. 0.124 1.014 .+-. 0.156 0.743 .+-. 0.083 0.245 .+-. 0.027 0.241
.+-. 0.038 control group Adjuvant 3.026 .+-. 0.505 1.023 .+-. 0.154
1.072 .+-. 0.150 0.750 .+-. 0.142 0.254 .+-. 0.045 0.266 .+-. 0.042
control group Low dosage 3.101 .+-. 0.346 1.028 .+-. 0.090 1.034
.+-. 0.198 0.765 .+-. 0.096 0.254 .+-. 0.027 0.254 .+-. 0.046 group
of the test sample High dosage 3.114 .+-. 0.283 1.038 .+-. 0.098
1.100 .+-. 0.215 0.779 .+-. 0.071 0.260 .+-. 0.024 0.275 .+-.
0.054* group of the test sample
Sperm motility and counts of the parent male rats: as compared with
the negative control group, no statistical differences of the sperm
motility, the ratio of forward motile sperm, the average speed, the
speed for linear motion, the speed for curvilinear motion, the
amplitude of lateral head displacement, the beat cross frequency,
the forward motility, the straight motility, the elongation and the
sperm counts of cauda epididymidis were observed in the male rats
in each dosage group of the test sample (P>0.05).
Morphological examination on the sperm of the parent male rats: as
compared with the negative control group, no statistical
differences of the headless deformity rate, the hook-less deformity
rate, amorphous head deformity rate, the tail-less deformity rate,
the folding deformity rate, the prehensile tail rate, the rate of
excessive hook bending and the total deformity rate of sperms were
observed in the animals in the high dosage group of the test sample
(P>0.05).
TABLE-US-00033 TABLE 33 Examination on the parent female rats by
cesarean delivery (conducted on GD20) Adjuvant Negative control
control group Low dosage group High dosage group group (3 of the
test sample of the test sample (0 dose/animal) doses/animal) (1
dose/animal) (3 doses/animal) Weight of uterus 75.218 .+-. 19.530
70.757 .+-. 23.323 73.536 .+-. 13.972 77.624 .+-. 12.000 and fetus
(g) Average number 18.5 .+-. 3.4 17.5 .+-. 3.6 17.6 .+-. 3.6 17.5
.+-. 3.0 of corpus luteum graviditatis Average 15.1 .+-. 3.5 14.2
.+-. 3.5 14.5 .+-. 2.8 15.3 .+-. 2.1 nidation number Nidation rate
82.63 .+-. 16.84 82.11 .+-. 16.94 83.93 .+-. 15.01 88.78 .+-. 11.99
(%) Loss rate before 17.37 .+-. 16.84 17.89 .+-. 16.94 16.07 .+-.
15.01 11.22 .+-. 11.99 nidation (%) Loss rate after 8.72 .+-. 11.26
9.88 .+-. 21.92 8.89 .+-. 9.75 7.21 .+-. 7.64 nidation (%) Live
birth 91.28 .+-. 11.26 90.12 .+-. 21.92 91.11 .+-. 9.75 92.79 .+-.
7.64 rate (%) Adsorbed 8.72 .+-. 11.26 9.88 .+-. 21.92 8.89 .+-.
9.75 6.95 .+-. 7.57 embryo rate (%) Stillbirth rate 0.00 .+-. 0.00
0.00 .+-. 0.00 0.00 .+-. 0.00 0.27 .+-. 1.25 (%)
TABLE-US-00034 TABLE 34 Examination on the fetal rats Gender ratio
(number of Fetal body Fetal tail male Fetal body weight (g) Weight
of length length rats/number of Groups Male Female placenta (g)
(mm) (mm) female rats) Negative 3.549 .+-. 0.314 3.451 .+-. 0.307
0.499 .+-. 0.065 36.47 .+-. 0.90 11.87 .+-. 0.34 1.3 (191/144)
control group Adjuvant 3.637 .+-. 0.301 3.516 .+-. 0.335 0.541 .+-.
0.183 36.76 .+-. 0.86 11.89 .+-. 0.41 1.0 (135/137) control group
Low dosage 3.653 .+-. 0.406 3.521 .+-. 0.303 0.499 .+-. 0.060 36.57
.+-. 0.97 12.00 .+-. 0.31 1.3 (181/139) group of the test sample
High dosage 3.614 .+-. 0.337 3.461 .+-. 0.375 0.476 .+-. 0.054
36.64 .+-. 0.99 12.03 .+-. 0.35 1.2 (171/142) group of the test
sample
As compared with the negative control group, no statistical
differences of the deformed fetal appearance rate and the abnormal
placenta rate were seen in each dosage group of the test sample
(P>0.05).
TABLE-US-00035 TABLE 35 Examination on the fetal skeleton Negative
High dosage group of control group the test sample (3 (0
dose/animal) doses/animal) Number of the litters examined 23 22
Number of the fetal rats 166 163 examined Left middle hand bones
3.41 .+-. 0.32 3.50 .+-. 0.33 Right middle hand bones 3.41 .+-.
0.32 3.48 .+-. 0.33 Left middle foot bones 3.98 .+-. 0.05 4.00 .+-.
0.02 Right middle foot bones 3.99 .+-. 0.05 4.00 .+-. 0.02 Number
of the breastbones 5.46 .+-. 0.56 5.42 .+-. 0.65 Number of the
sacrococcygeal 7.33 .+-. 0.46 7.36 .+-. 0.68 vertebra Rate of
abnormal bones (%) 5.21 .+-. 10.23 2.24 .+-. 4.91 Rate of aberrant
bones (%) 14.72 .+-. 18.28 11.02 .+-. 17.53
TABLE-US-00036 TABLE 36 Examination on the fetal organs Negative
High dosage group of control group the test sample (3 (0
dose/animal) doses/animal) Number of the litters examined 24 22
Number of the fetal rats 161 150 examined Rate of abnormal organs
(%) 0.60 .+-. 2.92 0.76 .+-. 3.55 Rate of aberrant organs (%) 27.41
.+-. 23.21 28.78 .+-. 24.12
Examination Items for the Fetal Rats of Generation F1 Survival of
the fetal rats of generation F1 Body weight and gender ratio of the
fetal rats of generation F1 Deformed appearance of the fetal rats
of generation F1 Examination on the body development indices of the
fetal rats of generation F1; Examination on the reflex development
indices of the fetal rats of generation F1; As compared with the
negative control group, no statistical differences of each index
were seen in the generation F1 fetal rats in each dosage group of
the test sample (P>0.05).
Results: No adverse effects were seen on the fertility of the
parent male and female rats, and on the gestation/lactation of the
female rats. No toxicity and teratogenicity were seen on the
development of the embryo-fetus. No obvious effects on the body
development indices and the reflex development indices were seen in
the fetal rats of generation F1.
D. Establishment of Animal Models
During the research process of the present invention, suitable
animal models were demanded to simulate human pathogenic
environment for investigation on the pathogenic mechanism and
process of Staphylococcus aureus. The action mechanism of
therapeutic medicines or vaccines could be elucidated by
pharmacological and pharmacodynamic experiments, so that novel
medicines for the prevention and treatment of Staphylococcus aureus
infections would be developed. Accordingly, animal models were
required to stimulate the diseases. In recent years, a great number
of experiments were carried out by investigators in China or abroad
using pneumonia animal models, including rhesus monkey model,
rabbit model, mouse model, pig model, and rat model, etc. The
monkey was an ideal experimental animal, due to similar physiologic
structure to human. However, the use of monkey in experiment was
limited due to lower quantity and higher cost. At present, rodents
were commonly adopted in a pneumonia animal model in China or
abroad.
Researches on establishing a pneumonia mouse model were reported.
For example, a pneumonia mouse model was generated by the follows
methods: adding 50 .mu.L bacteria suspension directly to the left
nasal cavity of a BALB/C mouse dropwise, as described by Wang Xing
et al. (Wang Xing et al., Chinese Journal of antibiotics, 2011,
8(36), 8, 617-602); or after weighing and intraperitoneal
anesthesia using 10% chloral hydrate solution, fixing a Kunming
mouse of 6-8 weeks old on an experiment table in supine position to
expose glottis, and then injecting bacteria suspension to the
tracheae using a self-made syringe with a blunt needle inserted
into the upper trachea, as described by Lv Xiaoyan et al. (Lv
Xiaoyan et al., Shandong Medical Journal, 2010, 50(3), 10-12). The
pneumonia model could be established by the methods above. However,
problems still existed in these methods, which mainly included: 1.
a proportion of bacteria suspension might be added into oral cavity
rather than lung for the mouse used without anaesthesia, since the
nasal cavity was in connection with the oral cavity, so that the
infection dosage could not be controlled precisely, resulting in an
unstable model; 2. as an invasive method, inserting a needle into
the trachea would hurt the organs of the animal, or even kill the
animal during operation, although the infection dosage could be
controlled; 3. there were so many mouse species that no susceptible
model had been definitely determined; 4. unstable infection and
colonization might lead to an unstable model, and success in
establishing a model depended on a stable infection dosage.
Based on the problems above, in this section, the present invention
provided a method for establishing a Staphylococcus aureus
pneumonia model, in order to enhance the accuracy of the infection
by bacteria suspension, increase the infection efficiency, and
improve the stability of the pneumonia model.
The method for establishing a Staphylococcus aureus pneumonia model
includes the following steps:
(1) anesthetizing the mouse; (2) subject the mouse to nasal
drip;
wherein:
In step (1), the mouse was anaesthetized by isoflurane; preferably,
anaesthesia was performed by inhalation of isoflurane at a
concentration of 4%-6% (preferably, 5%) delivered in oxygen, and
after the stage of deep anaesthesia was attained, anaesthesia was
maintained by delivering isoflurane at a concentration of 2%-4%
(preferably, 3%).
In step (2), a. during nasal drip, the supination amplitude of the
mouse head should not larger than the physiological curvature of
nasopharynx; b. medicine solution was dropped along the lateral
wall of nasal cavity, in order to avoid as many bubbles generated
during nasal drip as possible; c. at the beginning moment of
inspiration, less than 5 .mu.L medicine was dropped; d. bacteria
density was from 2.times.10.sup.10-2.times.10.sup.11 CFU/mL; e
total dosage of bacteria was 5.times.10.sup.9-1.times.10.sup.10 CFU
during nasal drip. Preferably, two separate nasal drips (each of a
volume of 25-35 .mu.L) were performed with a total volume of 50-70
.mu.L and a interval of 30 min.
Preferably, infection was evaluated in two ways: lung tissue
culture and pathological section. Healthy mouse was used as a
control.
Preferably, C57BL/6J mouse was used; and more preferably, the
C57BL/6J mouse was of SPF grade and 8-week old.
The infection conducted by the method of the invention was easily
controllable, and stable, with higher infection rate. The infection
could result in obvious symptoms of pneumonia.
The materials and primary reagents used in this section were as
follows:
1. Experiment Animals
Female C57BL/6J mice of 6-8 weeks old, supplied by Beijing HFK
Bioscience Co., Ltd.
2. Strains (Purchased from ATCC);
Staphylococcus aureus strain MRSA 252, Staphylococcus aureus strain
MRSA US-300, Staphylococcus aureus strain MRSA MW2, Staphylococcus
aureus strain MRSA WHO-2, Staphylococcus aureus strain MRSA COL,
and Staphylococcus aureus strain MRSA NEWMAN, were preserved in a
refrigerator at -80.degree. C. in our lab.
3. Equipments and Reagents
All equipments used were ordinary in the field of biology,
including a incubator, a centrifuge, a spectrophotometer, a
super-clean bench, and a shaker, etc.
MHA culture medium (purchased from Beijing Aoboxing Biotechnology
Co., Ltd.), MHB culture medium (purchased from Beijing Aoboxing
Biotechnology Co., Ltd.), MH agar plate and liquid medium
(purchased from Beijing Aoboxing Biotechnology Co., Ltd.), NaCl
(NS, supplied by Southwest Pharmaceutical Co., Ltd.), and
isoflurane (purchased from Shandong Keyuan Pharmaceutical Co.,
Ltd.).
Recombinant Staphylococcus aureus vaccine, containing SpA5, HI,
MntC and mSEB protein, each at a concentration of 50 .mu.g/ml.
Example 33: Preparation of Staphylococcus aureus Suspension
Cryopreserved MRSA 252 was revived by aerobic culture on an MH agar
plate at 37.degree. C. over night. Individual colonies were picked
up and inoculated to 1000 mL MH liquid medium. Subsequently, it was
incubated aerobically in a shaker at 210 rpm for 7 h at 37.degree.
C. The suspension was centrifugated at 4700 rpm to collect the
bacteria, which was then re-suspended in physiological saline after
washing by physiological saline twice. OD.sub.600 of the test
suspension was detected by a spectrophotometer, and the
concentration of bacteria was calculated based on
1OD=2.times.10.sup.9 CFU/mL.
Example 34: Establishment of Anaesthesia and Nasal Drip Scheme of
Mouse
In the infection test using the Staphylococcus aureus pneumonia
model, mice were initially anaesthetized by isoflurane to ensure a
successful nasal drip. Procedure for anaesthesia: anaesthesia was
performed by inhalation of isoflurane at a concentration of 5%
delivered in oxygen, and after the stage of deep anesthesia was
attained, anaesthesia was maintained by delivering isoflurane at a
concentration 3%. Using this method, favorable anaesthesia effect
could be achieved. A stable model depended on a controlled dosage
of nasal drip. In order to control the dosage better, to prevent
the bacteria suspension from entering the oral cavity, and to avoid
generating bubbles, the following measures were adopted: the
supination amplitude of the mouse head should not be larger than
the physiological curvature of nasopharynx during nasal drip, since
the physiological curvature allowed the nasal drops to enter the
oral cavity more easily at larger supination amplitude; (2)
dropping the medicine solution along the lateral wall of nasal
cavity, in order to avoid as many bubbles generated during nasal
drip as possible; (3) performing with a good rhythm, and dropping
less than 5 .mu.L medicine at the beginning moment of inspiration;
(4) two separate nasal drips (each of a volume of 25 .mu.L) were
performed at an interval of 30 min, since the dosage of 50 .mu.L
used in this Example was too large for a single dose. (5) As
discovered according to the experiments using the bacteria
suspensions of various concentrations, less bubbles were generated
at a bacteria density of about 2.times.10.sup.10-2.times.10.sup.11
CFU/ml.
Example 35: Preliminary Investigation on the Infection Dosage
The MRSA 252 suspension of Examples 33 was adjusted to 5
concentrations using physiological saline. Female C57BL/6J mice
were randomly divided into 5 groups. After anaesthesia by
isoflurane, infection was performed to the mice via nasal drip with
an infection dosage of 50 .mu.L for each mouse. Physiological
saline (NS) of the same volume was used as a control. Animal Groups
and infection dosages were listed in Table 37. After infection,
mice were observed every other day for survival or death. The
observation continued for 10 days. After the observation, the rest
animals were euthanized by CO.sub.2 inhalation.
TABLE-US-00037 TABLE 37 Preliminary investigation on the infection
dosage in the Staphylococcus aureus pneumonia model Dropping Dosage
Concentration volume SA strains Groups (CFU) Number (CFU/ml)
(.mu.L) MRSA 252 1 1.5 .times. 10.sup.10 10 3 .times. 10.sup.11 50
MRSA 252 2 1.2 .times. 10.sup.10 10 2.4 .times. 10.sup.11 50 MRSA
252 3 1 .times. 10.sup.10 10 2 .times. 10.sup.11 50 MRSA 252 4 8
.times. 10.sup.9 10 1.6 .times. 10.sup.11 50 MRSA 252 5 5 .times.
10.sup.9 10 1 .times. 10.sup.11 50
Initially, C57BL/6J mice were infected by the suspensions of
Staphylococcus aureus strain MRSA 252 at various concentrations,
and after 10 days observation, as shown in FIG. 55, the results
were as follows: only 20% of mice died at a infection dosage of
5.times.10.sup.9 CFU, 80% died at 1.times.10.sup.10 CFU, and no
obvious change was observed with further increased dosages, when
infected by nasal drip.
Example 36: Determination of Final Infection Dosage
Based on preliminary investigation, the concentration was further
adjusted. After anaesthesia by isoflurane, female C57BL/6J mice
were infected via nasal drip with an infection dosage of 50 .mu.L
for each mouse. Physiological saline (NS) of the same volume was
used as a control. Animal Groups and infection dosages were listed
in Table 38. After infection, mice were observed every other day
for survival or death. The observation continued for 10 days. After
the observation, the rest animals were euthanized by CO.sub.2
inhalation.
TABLE-US-00038 TABLE 38 Final infection dosage for the
Staphylococcus aureus pneumonia model Dropping Dosage Concentration
volume SA strains Group (CFU) Number (CFU/ml) (.mu.L) MRSA 252 1
.sup. 1 .times. 10.sup.10 10 2 .times. 10.sup.11 50 MRSA 252 2 8
.times. 10.sup.9 10 1.6 .times. 10.sup.11 50 MRSA 252 3 5 .times.
10.sup.9 10 1 .times. 10.sup.11 50
Based on preliminary investigation, the concentration of bacteria
for infection was further adjusted. The highest concentration of
bacteria was controlled at 2.times.10.sup.11 CFU/ml, since higher
concentration is difficult to be conducted for nasal drip. After
observation, as shown in FIG. 56, the results were listed as
follows: the mortality rate was 20% at an infection dosage of
5.times.10.sup.9 CFU, 40% at an infection dosage of
8.times.10.sup.9 CFU, and 90% at an infection dosage of
1.times.10.sup.10 CFU, which was substantially consistent with the
previous results. The dosage of nasal drip was finally determined
as 1.times.10.sup.10 CFU for mouse.
Example 37: Evaluation on Infection
10 female C57BL/6J mice were infected at a final dosage of
1.times.10.sup.10 CFU. The mice were killed 12 h after infection.
Subsequently, both lungs were washed by physiological saline. The
lungs were ground by adding 1 mL physiological saline. 100 .mu.L
suspension obtained after grinding was diluted in a ratio of
1:10000000. 100 .mu.L dilution was smeared on an MH plate. After
aerobic culture over night, the plate was observed and the colonies
were counted. Log 10 of the colony counts was plotted. The mice
were killed by cervical dislocation after anaesthesia, at 72 h
after infection. Both lungs of the mouse were placed in 10% neutral
formalin solution, fixed overnight, embedded in paraffin, sectioned
(30 .mu.m), and HE stained. Meanwhile, nasal drip was performed to
10 female C57BL/6J mice using physiological saline as a negative
control.
The mice were killed 12 h after infection. The lung tissue was
cultured, and the result was shown in FIG. 57. After MRSA 252
infection, a positive rate of 100% was obtained for Staphylococcus
aureus culture of the lung tissue, whereas negative result was
observed for the control group, demonstrating that the infection
was successful and stable.
The mice were killed 72 h after infection. Both lungs of the mouse
were placed in 10% neutral formalin solution, fixed overnight,
embedded in paraffin, sectioned (30 .mu.m), and HE stained. The
result indicated that in mice of the experimental group, the
following symptoms were observed, including: disappearance of some
pulmonary alveolus structure, hyperemia and massive inflammatory
cell infiltration. FIG. 58 showed thickening of alveolar septa,
vicarious swelling of pulmonary alveolus and massive inflammatory
cell infiltration. Obvious symptoms of pneumonia were induced in
the Staphylococcus aureus infected C57BL/6J mouse model prepared
using this method.
Example 38: Evaluation on the Immunoprotective Effect of the
Recombinant Staphylococcus aureus Vaccine in a Pneumonia Model
C57BL/6J mice were immunized by intramuscular injection of the
recombinant Staphylococcus aureus vaccine using the procedure of
immunization on Day 0, Day 3 and Day 7. On Day 10-14 after the last
immunization, the cryopreserved MRSA 252, MRSA US-300, MRSA MW2,
MRSA WHO-2, MRSA COL, and MRSA NEWMAN strain were revived. The
concentration of the bacteria suspension was adjusted to LD80-LD90.
After anaesthesia by isoflurane, infection was performed to the
mice via nasal drip with an infection dosage of 50-70 .mu.L for
each mouse. Physiological saline (NS) of the same volume was used
as a control. Animal Groups and infection dosages were listed in
Table 39.
TABLE-US-00039 TABLE 39 Various strains and infection dosages used
in the Staphylococcus aureus pneumonia model Concen- Volume Dosage
tration of nasal SA strains Group (CFU) Quantity (CFU/ml) drip
(.mu.l) MRSA 252 NS 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11
60 MRSA 252 vaccine 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11
60 MRSA COL NS 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11 60
MRSA COL vaccine 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11 60
MRSA WHO-2 NS 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11 60 MRSA
WHO-2 vaccine 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11 60 MRSA
MW2 NS 8 .times. 10.sup.9 10 1.6 .times. 10.sup.11 50 MRSA MW2
vaccine 8 .times. 10.sup.9 10 1.6 .times. 10.sup.11 50 MRSA US-300
NS 1.12 .times. 10.sup.10 10 1.6 .times. 10.sup.11 70 MRSA US-300
vaccine 1.12 .times. 10.sup.10 10 1.6 .times. 10.sup.11 70 MRSA NS
9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11 60 NEWMAN MRSA
vaccine 9.6 .times. 10.sup.9 10 1.6 .times. 10.sup.11 60 NEWMAN
After infection, mice were observed every other day for survival or
death. The observation continued for 10 days. After the
observation, the rest animals were euthanized by CO.sub.2
inhalation.
TABLE-US-00040 TABLE 40 Survival rate (10 days later) in the
pneumonia model infected by various strains after immunization by
the Staphylococcus aureus vaccine Number of Mortality Survival SA
strain Group Number dead mice rate rate MRSA 252 NS 10 9 90% 10%
MRSA 252 vaccine 10 2 20% 80% MRSA COL NS 10 9 90% 10% MRSA COL
vaccine 10 3 30% 70% MRSA WHO-2 NS 10 10 100% 0% MRSA WHO-2 vaccine
10 4 40% 60% MRSA MW2 NS 10 7 70% 30% MRSA MW2 vaccine 10 1 10% 90%
MRSA US-300 NS 10 8 80% 20% MRSA US-300 vaccine 10 2 20% 80% MRSA
NEWMAN NS 10 8 80% 20% MRSA NEWMAN vaccine 10 2 20% 80%
As shown, the mice can be effectively infected by the method of the
invention, and an animal pneumonia model infected by Staphylococcus
aureus was successfully established. The mouse model was stable
after infection, and suitable for researches, such as evaluation on
the immunoprotection by the Staphylococcus aureus vaccine.
E. Dissociation of the Vaccine Formulation and Method for Antigen
Content Detection
During the research and development of the recombinant protein
vaccines, the stability of antigen protein after adsorption by the
adjuvant should be monitored, i.e., the antigen content in the
finished vaccine product must be precisely determined. However, a
finished vaccine product was typically a colloidal complex formed
by adsorption of the antigen protein by the adjuvant. Accordingly,
the difficulties that should be addressed for antigen content
detection lied in how to make the protein dissociate from the
adjuvant while retain its properties. At present, methods for
dissociation between the protein and the adjuvant have been
reported by using sodium citrate, or guanidinium hydrochloride,
etc. However, problems, such as incomplete dissociation, and longer
dissociation duration, etc., were often accompanied. Additionally,
SDS-PAGE, ELISA, or HPLC etc would be commonly used for detecting
the antigen content in a finished product of protein vaccine after
dissociation between the antigen and the adjuvant. However, in the
recombinant Staphylococcus aureus vaccine of the invention, the
antigen components were complicated with similar molecular weights
for several antigen proteins, so that the protein bands or peaks
might overlap each other when detected by SDS-PAGE or HPLC,
respectively, which would lead to inaccurate quantification.
Furthermore, monoclonal antibodies for all 4 corresponding proteins
would be required when detected by ELISA, resulting in higher cost
and extended research period.
In term of the problems above, after intensive investigation,
dissociation between the antigen protein and the adjuvant was
implemented by the inventors in 10 min using a sodium carbonate
solution. Meanwhile, western blot was performed for antigen
standards of different concentrations and for the samples of the
finished vaccine products after dissociation at the same time using
the rabbit anti-serums against these 4 antigens; after development
by enzymatic reaction, the grey-scale values for each band were
detected by a scanner; a standard curve was established by
correlating the grey-scale values to the antigen concentrations;
the antigen contents in the finished product were calculated
according to the regression equation. These methods could be used
for quantification of an antigen in a finished vaccine product, and
had advantages, such as easy operation, and good repeatability. At
present, no related research has been reported yet.
In this section, the present invention provided a dissociation
method for the Staphylococcus aureus vaccine formulation and a
method for antigen content detection, which includes:
1) the dissociation solution: 1.5-2.5 M sodium carbonate
solution
Method for dissociation: the dissociation solution was combined
with the vaccine solution in a volume ratio of 0.5-2:1; and
preferably, the concentration of the dissociation solution was 2 M,
and the volume ratio between the dissociation solution and the
vaccine solution was 1:1.
Taking the 4-component recombinant Staphylococcus aureus vaccine of
the invention as an example, a dose of the finished vaccine product
was centrifugated at 5000 rpm for 10 min at room temperature.
Subsequently, a half volume of the supernatant was discarded, and
the dissociation solution of the same volume was added. The mixture
was then shaken at room temperature for 10 min. For example, if 600
.mu.L of the finished vaccine product was taken out, 300 .mu.L of
the supernatant was discarded and another 300 .mu.L of the
dissociation solution was added.
2) detection of the antigen content: western blot was performed for
antigen standards of different concentrations and for the samples
of the finished products after dissociation at the same time using
the anti-serums against various antigens; after development by
enzymatic reaction, the grey-scale values for each band were
detected by a scanner; a standard curve was established by
correlating the grey-scale values to the antigen concentrations;
the antigen contents in the finished product were calculated
according to the regression equation.
Preferably, the finished vaccine product comprises the antigen and
the adjuvant.
Preferably, the finished vaccine product comprises a plurality of
antigens, such as 2, 3, 4, 5, or 6 antigens. More preferably, the
antigens were selected from SpA5 protein, HI protein, mSEB protein,
and MntC protein.
In another aspect, the present invention provided a kit for the
method of the invention, which comprises: a dissociation solution
and reagents for detecting the antigen content. Preferably, the
dissociation solution was 1.5-2.5 M sodium carbonate solution; and
more preferably, the dissociation solution was 2 M sodium carbonate
solution; the reagents for detecting the antigen content were the
reagents for western blot.
Using this method, rapid and complete dissociation between the
antigen and the adjuvant could be achieved. Meanwhile, complex
antigens in a finished vaccine product could be quantified.
Accordingly, problems in the vaccine formulation assay, such as the
dissociation and the antigen content detection in a finished
product, were solved. The present invention was applicable to the
dissociation and antigen content detection for the protein vaccines
in the bio-pharmaceutical field.
The antigen proteins and various reagents used in this section were
listed as follows:
1) Materials
Stock solutions of 4 antigens in the recombinant Staphylococcus
aureus vaccine of the invention (SpA5 (KKAA), HI, MntC, and mSEB),
with a concentration for each solution of 50 .mu.g/ml;
In the finished 4-component recombinant Staphylococcus aureus
vaccine product of the invention, the concentration for each
component was 50 .mu.g/ml;
Rabbit polyantiserums against 4 antigen components: New Zealand big
ear white rabbits were immunized by the finished recombinant
Staphylococcus aureus vaccine product based on the procedure of
immunization on D0, D14, and D21 for 3 times, and the serums were
obtained by blood collection on Day 14 after the last
immunization.
2) Equipments
Water purification system (ELGA), electronic balance (Mettler
Toledo), electrophoresis gel mould (BIORAD), protein gel imaging
system (BIORAD), electrophoresis apparatus (BIORAD) and Semi-dried
gel transfer apparatus (BIORAD).
3) Reagents
Trihydroxymethyl aminomethane (Tris), glycine, methanol, sodium
chloride, hydrochloric acid, Tween 20, skimmed milk powder, DAB
color developing reagent kit, 30% acrylamide, ammonium persulfate,
1.5 M Tris-HCl (pH 8.8), 10% SDS, 10% ammonium persulfate, TEMED,
1.0 M Tris-HCl (pH 6.8), 5.times. loading buffer, PVDF membrane
(Milipore), and horse radish peroxidase-labled goat anti-rabbit IgG
secondary antibody (BD corporation).
4) Preparation of Reagents
Dissociation solution: 2 M sodium carbonate solution;
Transfer buffer: 3.03 g Tris and 14.41 g glycine were added to 200
mL methanol, to which water was added to a final volume of 1 L. The
solution was stored at 4.degree. C.
10.times.TBS buffer: 24.228 g Tris and 87.75 g sodium chloride were
dissolved in a suitable amount of water, and the pH value was
adjusted to 7.5 using HCl, to which water was added to a final
volume of 1 L. The solution was stored at 4.degree. C.
TBST washing solution: 10.times.TBS buffer was diluted to
1.times.TBS buffer, to which 0.5% Tween 20 was added to prepare
1.times.TBST washing solution.
10.times.SDS-PAGE electrophoresis buffer: 3.03 g Tris, 14.41 g
glycine and 1 g SDS were dissolved in ddH.sub.2O, followed by
adding ddH.sub.2O to a final volume of 1 L.
Other solutions were prepared as described herein elsewhere.
Example 39: Determination of the Dissociation Duration Between the
Antigen and the Adjuvant
Taking the recombinant Staphylococcus aureus vaccine as an example,
600 .mu.l of the finished vaccine product in one dose was
centrifugated at 5000 rpm for 10 min at room temperature.
Subsequently, 300 .mu.L of the supernatant was discarded, and
subsequently 300 .mu.L of the dissociation solution (2 M
Na.sub.2CO.sub.3) was added. The mixture was then suspended at room
temperature, and shaken for 10 min, 20 min and 30 min. After
standing for 5 min, 40 .mu.L supernatant was added to 10 .mu.L
5.times. loading buffer, and subjected to SDS-PAGE. As shown in
FIG. 59, after dissociation for 10 min, 20 min and 30 min, bands
appeared on the gel with similar size, in a sequence from top to
bottom of HI (48.2 kDa), MntC (32.9 kDa), SpA5 (32.7 kDa), and mSEB
(28.1 kDa). The result indicated that there was no significant
difference among various dissociation times. Accordingly, the
dissociation time was determined as 10 min.
Example 40: Comparison Between the Dissociation Method of the
Invention and Other Methods
The dissociation method of Example 39 was adopted in the present
invention, with a dissociation duration of 10 min. The same
procedure as described above was used for the control method,
except for a varied formulation of the dissociation solution and
different dissociation duration. There were 3 control methods,
specifically as follows: (1) dissociation by 1 M sodium citrate at
room temperature for 1 h; (2) dissociation by 2 M guanidinium
hydrochloride at room temperature over night; (3) dissociation by
0.1 M citric acid at room temperature for 1 h. As shown in FIG. 60,
more clear bands appeared on the gel for the sample dissociated by
sodium carbonate, as compared with other methods, suggesting a more
complete dissociation by this method. Additionally, what's more
important was that the dissociation duration for the method of the
invention was short, with simple operation. As indicated by these
results, the method of the invention has more advantages over the
reported methods.
Example 41: Detection of Antigen Content in a Finished Vaccine
Product
Methods:
Preparation of the samples for electrophoresis: suitable amount of
the stock antigen solution was diluted by ddH.sub.2O to a
concentration of 10 .mu.g/ml, 30 .mu.g/ml, 50 .mu.g/ml, 70
.mu.g/ml, and 90 .mu.g/ml, to which a certain amount of 5.times.
loading buffer was added to obtain 1.times. loading buffer. The
samples were heated at 100.degree. C. for 5 min. At the same time,
one dose of finished vaccine product was taken, and 600 .mu.L of
the finished vaccine product was withdrawn to a 1.5 mL EP tube
after mixed, and centrifugated at 5000 rpm for 10 min. 300 .mu.L of
the supernatant was discarded, and 300 .mu.L 2 M Na.sub.2CO.sub.3
solution was added, and mixed. After becoming clear, the solution
was centrifugated. Subsequently, to a suitable amount of
supernatant, 5.times. loading buffer was added. The samples were
then heated at 100.degree. C. for 5 min.
SDS-PAGE: 20 .mu.l samples of various antigen concentrations were
loaded. The samples initially run at 80 v for 20 min, and then at
160 v until bromphenol blue was 0.5 cm away from the edge of glass
plate.
Transfer: 2 pieces of filter paper was soaked in the transfer
buffer, and a PVDF membrane was wetted by methanol. Transfer was
conducted using a semi-dried gel transfer apparatus operated at 20
v for 20 min.
Blotting: after transfer, the PVDF membrane was washed by the TBST
washing solution for 3 times, each for 10 min. The PVDF membrane
was then placed in the blocking solution at 4.degree. C. over
night. Subsequently, the PVDF membrane was washed by the TBST
washing solution for 3 times, each for 10 min. The self-made
primary antibody (rabbit polyclonal antibody) was diluted in
1.times.TBS buffer for 5000 folds. The blocked PVDF membrane was
then incubated in the primary antibody at 37.degree. C. for 1 h.
Subsequently, the PVDF membrane was washed by the TBST washing
solution for 3 times, each for 10 min. The horse radish
peroxidase-labeled goat anti rabbit secondary antibody was diluted
in 1.times.TBS buffer for 5000 folds. Subsequently, the PVDF
membrane was incubated in the secondary antibody at 37.degree. C.
for 40 min. The PVDF membrane was then washed by the TBST washing
solution for 4 times, each for 10 min. Color developing was carried
out according to the instructions of the DAB color developing
reagent kit. After suitable developing, the reaction was stopped by
washing with water. Evaluation on the results: the bands for the
antigen standards should gradually become thicker. The grey-scale
value was detected using a gel imaging system to establish a
standard curve. The content of each antigen in the finished vaccine
product was determined.
Results: antigen standards of various concentrations (10 .mu.g/ml,
30 .mu.g/ml, 50 .mu.g/ml, 70 .mu.g/ml, and 90 .mu.g/ml) were used
as the control for western blot, as shown in FIG. 61. After an
obvious color development reaction with the rabbit polyclonal
antibody prepared using various antigens, a clear color band
appeared at the position of corresponding molecular weight. With
increased concentrations of each antigen, a clear size gradient was
observed for the band.
After scanning using a gel imaging system, the gray-scale values
for each band were obtained, as listed in Table 41. Additionally, a
standard curve was established between the gray-scale values and
the antigen concentrations, as shown in FIG. 62. The results
indicated that a clear linear correlation was observed between the
gray-scale values of the western blot bands and their
concentrations for each antigen standard, with a correlation
coefficient R.sup.2 larger than 0.98, which could be used for the
antigen content detection in the finished vaccine product.
TABLE-US-00041 TABLE 41 Results of the gray-scale values of the
western blot bands for different antigen standards Antigen
concentration (.mu.g/ml) HI mSEB MntC SpA5 10 5553 4755 7375 7343
30 6645 6780 11442 11854 50 8281 8648 13910 13952 70 10151 9798
17071 17127 90 12571 11169 19033 19242
Using the same method, the gray-scale values of the western blot
bands were determined for the dissociated samples of the finished
vaccine product, and the contents of 4 antigens, including HI,
mSEB, MntC, and SpA5, were determined as 46.03 .mu.g/ml, 49.87
.mu.g/ml, 48.82 .mu.g/ml and 46.72 .mu.g/ml, respectively, based on
the equation in FIG. 62. The determined contents were close to
their actual content of 50 .mu.g/ml, and were within normal limits.
In another aspect, the results also demonstrated that a complete
dissociation could be implemented between the protein and the
adjuvant using the established method of the invention. The above
results also demonstrated that the antigen content in the finished
vaccine product could be determined by the standard curve
established using the gray-scale values of the western blot
bands.
TABLE-US-00042 TABLE 42 Detection of the contents of 4 antigens in
the finished vaccine product Type of antibody HI mSEB MntC SpA5
Gray-scale value of the 8292 8220 13596 13427 dissociated vaccine
sample Antigen content (.mu.g/ml) 46.03 49.87 48.82 46.72
F. Detection of the Bactericidal Antibody in the Serum
Example 42: Detection of the Antibody Serum-Mediated In Vitro
Opsono-Phagocytosis Capacity of Staphylococcus aureus in the Rat
Immunized by the Recombinant Staphylococcus aureus Vaccine
An antibody bound to IgG FcR at the surface of the neutrophilic
granulocyte through the Fc fragment of IgG (IgG1 and IgG3), and the
Fc fragment of IgM bound to IgM FcR at the surface of the phagocyte
by activating the complement system, which promoted the endocytosis
and killing capacity of neutrophilic granulocyte and phagocyte
against particulate antigens, such as bacteria etc.
1. Source of Antibody:
the rat serum of Example 30
2. List of Equipments
Incubator, superclean bench, low-speed centrifuge, low temperature
refrigerated centrifuge, protein purification system, ultra-low
temperature freezer, high & low temperature shaker (Thermo,
Ilcermo 481), nucleic acid and protein analyzer (Smart Spec.TM.
plus BD); OD tube (BD); and refrigerated centrifuge (Thermo
scientific, SORVAIL ST40R Centrifuge).
3. Reagents
MH (A) bacteria culture plate; physiological saline; IMDM medium;
fetal bovine serum (Gibco); Ca.sup.2+, or Mg.sup.2+ containing
Hanks's solution (Gibco); Ca.sup.2+, or Mg.sup.2+ free Hanks's
solution; RPMI-1640 culture solution (Gibco); dimethyl formamide
(DMF); sterilized water; and PBS.
4. Procedure
4.1 Culture and Differentiation of HL-60 Cells (Human Promyelocytic
Leukemia Cells, Supplied by Institute of Biochemistry and Cell
Biology, SIBS, CAS)
HL-60 cells stably grew in IMDM (Gibco) culture medium containing
20% fetal bovine serum in a disposable cell culture bottle. Mature
HL-60 cells were cultured at a concentration of 4.times.10.sup.5
cell/mL in complete medium (IMDM+20% FBS) containing 0.8% DMF for
differentiation for 4 days.
4.2 Preparation of the Working Seed Lot of MRSA252:
4.2.1 Culture and Cryopreservation of MRSA252
The bacteria were inoculated on an MHA solid plate by the
tri-linear method, labeled, and incubated at 37.degree. C. over
night. On the next day, single colony was picked up from the MHA
solid plate, and inoculated to a flask containing 20 mL MHB medium.
The flask was numbered and labeled, and placed in a shaker
controlled at temperature of 37.degree. C. shaken at a rotation
rate of 220 rpm. Enlarged cultivation was conducted for 6 h. After
detecting the concentration, bacteria were preserved by combining
the bacteria suspension with 50% glycerol in a ratio of 1:1, and
stored at -70.degree. C.
4.2.2 Determination of the Working Dilution of the Cryopreserved
Bacteria
500 .mu.L cryopreserved working seed of MRSA252 was thawed in a
water bath at 37.quadrature. and washed twice by the
opsonophagocytosis buffer (Hanks's solution containing Ca.sup.2+
and Mg.sup.2+), and centrifugated at 12000 rpm for 2 min. The
bacteria were suspended in 500 .mu.l opsonophagocytosis buffer, and
diluted in a series of eight 5-fold dilutions. 10 .mu.l suspension
was collected from each dilution, added to 8 individual wells of an
U-bottom 96-well plate (containing 20 .mu.l opsonophagocytosis
buffer in each well), and cultured in a shaker at 700 rpm for 30
min.
40 .mu.l of the differentiated HL-60 cell (concentration of
4.times.10.sup.5 cell/ml), pre-washed twice by Ca.sup.2+ and
Mg.sup.2+ containing Hanks's solution and Ca.sup.2+ and Mg.sup.2+
free Hanks's solution, was added to each well, and subsequently, 10
.mu.l complement was added. The mixture was cultured at 37.degree.
C. in an atmosphere of 5% CO.sub.2 in a shaker at 700 rpm for 45
min, and then placed in an ice bath for 20 min. Finally, it was
smeared on an MHA plate. Based on the result of pre-experiment, an
ideal colony count (120 CFU/Spot), i.e., the 5.sup.th dilution, was
selected as the working dilution.
4.3 Complement Collection and Screening:
The complement was harvested from the blood collected from the
heart of newborn chinchilla rabbits (Experiment Animal Center,
Third Military Medical), and stored at -70.degree. C.
The complement stored at -70.degree. C. was thawed at room
temperature, deactivated in a water bath at 56.degree. C. for 30
min, and stored at 4.degree. C. for further use. Differentiated
HL-60 cells were washed twice by the Ca.sup.2+ and Mg.sup.2+
containing Hanks's solution and the Ca.sup.2+ and Mg.sup.2+ free
Hanks's solution (centrifugation at 1200 rpm for 5 min). A suitable
amount of opsonophagocytosis buffer was added to a final cell
concentration of 1.times.10.sup.7 cell/ml, and the suspension was
stored at root temperature for further use.
Cryopreserved working seed of MRSA252 was diluted in a series of
eight 5-fold dilutions. At the same time, the same batch of
cryopreserved complement was thawed at room temperature. 10 .mu.l
of the complement was each added to the first 8 wells, to which 40
.mu.l of the differentiated HL-60 cell was added. Subsequently, to
the last 8 wells, 10 .mu.l of the deactivated complement and 40
.mu.l of the differentiated HL-60 cells were added, and cultured at
37.degree. C. in an atmosphere of 5% CO.sub.2, at 700 rpm for 45
min. After maintained in an ice bath for 20 min, the samples were
spotted. On the next day, the survival colonies were counted, and
non-specific killing (NSK) of this batch of complement was
calculated. Non-specific killing of the complement (%)=[Control
B-Control A)/Control A].times.100%. Wherein, Control A was the
survival colony counts after incubation of MRSA with the
heat-inactivated complement and HL-60 cells for 45 min, and Control
B was the survival colony counts after incubation of MRSA with the
complement and HL-60 cells for 45 min. Based on the experiment
results, the non-specific killing of this batch of complement was
39%, which met the requirements of the experiment.
4.4 Opsonophagocytosis Test:
4.4.1 All test serums were deactivated in a water bath at
56.degree. C. for 30 min, with 20 .mu.l in each well. For the
control well, the serum was substituted by 20 .mu.l
opsonophagocytosis buffer. To each well, 40 .mu.l of the HL-60
cells, 10 .mu.l of the complement, and 10 .mu.l of the bacteria
suspension were added and cultured at 37.degree. C. in an
atmosphere of 5% CO.sub.2 at 700 rpm for 45 min. After maintained
in an ice bath for 20 min, 10 .mu.l suspension was collected and
smeared on a plate. On the next day, the survival colonies were
counted.
4.4.2 Counting: the colonies on the MHA plate were counted.
4.4.3 Statistics: the bactericidal rate by each antibody was
calculated using the average colony counts on 3 plates. T test was
adopted for comparison among groups.
5. Result Evaluation:
For the control group, the result was the survival colony counts
after incubation of MRSA 252 with the complement and HL-60
cells.
For the experiment group, the result was the survival colony counts
after incubation of MRSA 252 with the antibody, the complement and
HL-60 cells. Bactericidal rate by the antibody=[(control
group-experiment group)/control group].times.100%
6. Results:
TABLE-US-00043 TABLE 43 Individual data of bacteria-killing
antibodies in the serum of rat immunized by repeated intramuscular
injection of the recombinant Staphylococcus aureus vaccine
Sterilizing rate of the antibody in the immunized serum Groups No.
D-1 D14 D21 D28 D57 Negative control 13-2481 -- -- died group
13-2482 -- -- -- 0.3% -- 13-2483 -- -- -- -- -- 13-2484 -- -- -- --
-- 13-2485 -- -- 0.1% -- 0.7% 13-2486 -- -- -- -- -- 13-2487 -- --
-- -- 0.7% 13-2488 -- -- -- -- -- 13-2489 -- -- -- -- -- 13-2490 --
-- -- 0.6% -- Low dosage 13-2491 -- 0.4% 7.4% 28.5% 41.2% group of
the 13-2492 -- 0.7% 1.8% 29.6% 33.5% test sample 13-2493 -- 1.8%-
6.8% 34.7% 46.0% 13-2494 -- -- 6.1% 19.8% 37.9% 13-2495 -- 3.9%
9.3% 34.7% 40.0% 13-2496 -- -- 3.7% 33.9% 34.5% 13-2497 -- -- 5.4%
27.5% 39.9% 13-2498 -- -- 9.5% 17.8% 26.3% 13-2499 -- 0.3% 5.8%
30.1% 41.1% 13-2500 -- -- 2.9% 28.9% 30.6% Middle dosage 13-2501 --
2.6% 27.3% 33.7% 57.2% group of the 13-2502 -- 3.2% 18.2% 29.9%
41.3% test sample 13-2503 0.4% 7.4% 21.7% 39.8% 60.4% 13-2504 --
2.8% 14.6% 53.1% 64.5% 13-2505 -- 3.0% 13.4% 27.9% 59.6% 13-2506 --
2.3% 20.1% 31.7% 64.8% 13-2507 -- 1.0% 25.2% 46.9% 50.3% 13-2508 --
2.5% 30.5% 46.5% 60.1% 13-2509 -- 3.3% 9.7% 28.8% 59.0% 13-2510 --
2.5% 16.3% 39.9% 47.6% High dosage 13-2511 -- 3.7% 20.0% 23.0%
39.8% group of the 13-2512 -- 4.1% 15.4% 41.9% 55.7% test sample
13-2513 -- 2.4% 32.1% 44.6% 46.0% 13-2514 0.3% 2.8% 25.5% 35.8%
52.4% 13-2515 -- 3.0% 11.9% 32.7% 61.3% 13-2516 0.2% 3.3% 17.7%
39.6% 57.8% 13-2517 -- 3.5% 34.3% 50.5% 53.6% 13-2518 -- 3.1% 9.9%
34.6% 48.2% 13-2519 -- 3.6% 28.3% 42.7% 49.3% 13-2520 -- 3.7% 37.4%
53.9% 64.0% Note: "--" represented a negative result of
neutralizing antibody detection.
The dosages and the animal numbering in this table was as described
in Table 18 of Example 30.
Apparent bactericidal activity was observed for the antibodies in
the serum of rat after immunization by the vaccine.
Example 43: Detection of the Antibody Serum-Mediated In Vitro
Opsono-Phagocytosis Capacity of Staphylococcus aureus in the
Cynomolgus Monkey Immunized by the Recombinant Staphylococcus
aureus Vaccine
Source of antibody: the cynomolgus monkey serum of Example 31;
The experiment method was the same as described in Example 42.
TABLE-US-00044 TABLE 44 Individual data of bacteria-killing
antibodies in the serum of cynomolgus monkey immunized by repeated
intramuscular injection of the recombinant Staphylococcus aureus
vaccine Sterilizing rate of the antibody in the serum Groups No.
D14 D28 Low dosage group of the 13-4177 13.1% 28.0% test sample
13-4178 11.7% 32.8% 13-4179 9.8% 21.3% 13-4180 17.4% 29.7% 13-4181
12.4% 19.5% 13-4182 16.3% 31.8% 13-4183 9.9% 22.9% 13-4184 13.2%
23.6% Middle dosage group of 13-4185 47.6% 60.4% the test sample
13-4186 52.1% 63.0% 13-4187 48.9% 66.3% 13-4188 48.7% 67.5% 13-4189
49.9% 68.o% 13-4190 51.3% 66.7% 13-4191 39.6% 59.7% 13-4192 50.8*
63.0% High dosage group of the 13-4193 55.3% 68.0% test sample
13-4194 47.8% 61.9% 13-4195 59.0% 67.4% 13-4196 55.5% 60.7% 13-4197
49.6% 63.9% 13-4198 60.0% 65.7% 13-4199 58.4% 69.9% 13-4200 53.3%
62.7% Note: "--" represented a negative result of bacteria-killing
antibody detection.
The dosages and the animal numbering in this table was as described
in Table 27 of Example 31.
Apparent bactericidal activity was observed for the antibodies in
the serum of cynomolgus monkey after immunization by the
vaccine.
SEQUENCE LISTINGS
1
161296PRTArtificial sequenceRecombinant SpA5(KKAA) protein 1Gly Pro
Leu Gly Ser Ala Gln His Asp Glu Ala Lys Lys Asn Ala Phe 1 5 10 15
Tyr Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly 20
25 30 Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn Val
Leu 35 40 45 Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys
Ala Asp Ala 50 55 60 Lys Lys Asn Lys Phe Asn Lys Asp Gln Gln Ser
Ala Phe Tyr Glu Ile 65 70 75 80 Leu Asn Met Pro Asn Leu Asn Glu Glu
Gln Arg Asn Gly Phe Ile Gln 85 90 95 Ser Leu Lys Ala Ala Pro Ser
Gln Ser Thr Asn Val Leu Gly Glu Ala 100 105 110 Lys Lys Leu Asn Glu
Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn 115 120 125 Lys Glu Lys
Lys Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu 130 135 140 Asn
Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro 145 150
155 160 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu
Ser 165 170 175 Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Lys
Lys Asn Ala 180 185 190 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn
Glu Glu Gln Arg Asn 195 200 205 Gly Phe Ile Gln Ser Leu Lys Ala Ala
Pro Ser Gln Ser Ala Asn Leu 210 215 220 Leu Ala Glu Ala Lys Lys Leu
Asn Asp Ala Gln Ala Pro Lys Ala Asp 225 230 235 240 Asn Lys Phe Asn
Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile Leu His 245 250 255 Leu Pro
Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu 260 265 270
Lys Ala Ala Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys 275
280 285 Leu Asn Asp Ala Gln Ala Pro Lys 290 295 2296PRTArtificial
sequenceRecombinant SpA5(RRAA) protein 2Gly Pro Leu Gly Ser Ala Gln
His Asp Glu Ala Arg Arg Asn Ala Phe 1 5 10 15 Tyr Gln Val Leu Asn
Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly 20 25 30 Phe Ile Gln
Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn Val Leu 35 40 45 Gly
Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala 50 55
60 Arg Arg Asn Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile
65 70 75 80 Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe
Ile Gln 85 90 95 Ser Leu Lys Ala Ala Pro Ser Gln Ser Thr Asn Val
Leu Gly Glu Ala 100 105 110 Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
Ala Asp Asn Asn Phe Asn 115 120 125 Lys Glu Arg Arg Asn Ala Phe Tyr
Glu Ile Leu Asn Met Pro Asn Leu 130 135 140 Asn Glu Glu Gln Arg Asn
Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro 145 150 155 160 Ser Gln Ser
Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser 165 170 175 Gln
Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Arg Arg Asn Ala 180 185
190 Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn
195 200 205 Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala
Asn Leu 210 215 220 Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala
Pro Lys Ala Asp 225 230 235 240 Asn Lys Phe Asn Lys Glu Arg Arg Asn
Ala Phe Tyr Glu Ile Leu His 245 250 255 Leu Pro Asn Leu Thr Glu Glu
Gln Arg Asn Gly Phe Ile Gln Ser Leu 260 265 270 Lys Ala Ala Pro Ser
Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys 275 280 285 Leu Asn Asp
Ala Gln Ala Pro Lys 290 295 3296PRTArtificial sequenceRecombinant
SpA5(KKVV) protein 3Gly Pro Leu Gly Ser Ala Gln His Asp Glu Ala Lys
Lys Asn Ala Phe 1 5 10 15 Tyr Gln Val Leu Asn Met Pro Asn Leu Asn
Ala Asp Gln Arg Asn Gly 20 25 30 Phe Ile Gln Ser Leu Lys Val Val
Pro Ser Gln Ser Ala Asn Val Leu 35 40 45 Gly Glu Ala Gln Lys Leu
Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala 50 55 60 Lys Lys Asn Lys
Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 65 70 75 80 Leu Asn
Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 85 90 95
Ser Leu Lys Val Val Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala 100
105 110 Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe
Asn 115 120 125 Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile Leu Asn Met
Pro Asn Leu 130 135 140 Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser
Leu Lys Val Val Pro 145 150 155 160 Ser Gln Ser Ala Asn Leu Leu Ala
Glu Ala Lys Lys Leu Asn Glu Ser 165 170 175 Gln Ala Pro Lys Ala Asp
Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala 180 185 190 Phe Tyr Glu Ile
Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn 195 200 205 Gly Phe
Ile Gln Ser Leu Lys Val Val Pro Ser Gln Ser Ala Asn Leu 210 215 220
Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp 225
230 235 240 Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile
Leu His 245 250 255 Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe
Ile Gln Ser Leu 260 265 270 Lys Val Val Pro Ser Val Ser Lys Glu Ile
Leu Ala Glu Ala Lys Lys 275 280 285 Leu Asn Asp Ala Gln Ala Pro Lys
290 295 4296PRTArtificial sequenceRecombinant SpA5(RRVV) protein
4Gly Pro Leu Gly Ser Ala Gln His Asp Glu Ala Arg Arg Asn Ala Phe 1
5 10 15 Tyr Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn
Gly 20 25 30 Phe Ile Gln Ser Leu Lys Val Val Pro Ser Gln Ser Ala
Asn Val Leu 35 40 45 Gly Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala
Pro Lys Ala Asp Ala 50 55 60 Arg Arg Asn Lys Phe Asn Lys Asp Gln
Gln Ser Ala Phe Tyr Glu Ile 65 70 75 80 Leu Asn Met Pro Asn Leu Asn
Glu Glu Gln Arg Asn Gly Phe Ile Gln 85 90 95 Ser Leu Lys Val Val
Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala 100 105 110 Lys Lys Leu
Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn 115 120 125 Lys
Glu Arg Arg Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu 130 135
140 Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Val Val Pro
145 150 155 160 Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu
Asn Glu Ser 165 170 175 Gln Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys
Glu Arg Arg Asn Ala 180 185 190 Phe Tyr Glu Ile Leu His Leu Pro Asn
Leu Asn Glu Glu Gln Arg Asn 195 200 205 Gly Phe Ile Gln Ser Leu Lys
Val Val Pro Ser Gln Ser Ala Asn Leu 210 215 220 Leu Ala Glu Ala Lys
Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp 225 230 235 240 Asn Lys
Phe Asn Lys Glu Arg Arg Asn Ala Phe Tyr Glu Ile Leu His 245 250 255
Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu 260
265 270 Lys Val Val Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys
Lys 275 280 285 Leu Asn Asp Ala Gln Ala Pro Lys 290 295
5516PRTStaphylococcus aureus 5Met Lys Lys Lys Asn Ile Tyr Ser Ile
Arg Lys Leu Gly Val Gly Ile 1 5 10 15 Ala Ser Val Thr Leu Gly Thr
Leu Leu Ile Ser Gly Gly Val Thr Pro 20 25 30 Ala Ala Asn Ala Ala
Gln His Asp Glu Ala Gln Gln Asn Ala Phe Tyr 35 40 45 Gln Val Leu
Asn Met Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe 50 55 60 Ile
Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly 65 70
75 80 Glu Ala Gln Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala
Gln 85 90 95 Gln Asn Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr
Glu Ile Leu 100 105 110 Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn
Gly Phe Ile Gln Ser 115 120 125 Leu Lys Asp Asp Pro Ser Gln Ser Thr
Asn Val Leu Gly Glu Ala Lys 130 135 140 Lys Leu Asn Glu Ser Gln Ala
Pro Lys Ala Asp Asn Asn Phe Asn Lys 145 150 155 160 Glu Gln Gln Asn
Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu Asn 165 170 175 Glu Glu
Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser 180 185 190
Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser Gln 195
200 205 Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala
Phe 210 215 220 Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln
Arg Asn Gly 225 230 235 240 Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser
Gln Ser Ala Asn Leu Leu 245 250 255 Ala Glu Ala Lys Lys Leu Asn Asp
Ala Gln Ala Pro Lys Ala Asp Asn 260 265 270 Lys Phe Asn Lys Glu Gln
Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 275 280 285 Pro Asn Leu Thr
Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys 290 295 300 Asp Asp
Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu 305 310 315
320 Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys
325 330 335 Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro
Gly Lys 340 345 350 Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp Gly Asn
Lys Pro Gly Lys 355 360 365 Glu Asp Asn Lys Lys Pro Gly Lys Glu Asp
Gly Asn Lys Pro Gly Lys 370 375 380 Glu Asp Gly Asn Lys Pro Gly Lys
Glu Asp Gly Asn Lys Pro Gly Lys 385 390 395 400 Glu Asp Gly Asn Lys
Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys 405 410 415 Glu Asp Gly
Asn Gly Val His Val Val Lys Pro Gly Asp Thr Val Asn 420 425 430 Asp
Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp Lys Ile Ala Ala Asp 435 440
445 Asn Lys Leu Ala Asp Lys Asn Met Ile Lys Pro Gly Gln Glu Leu Val
450 455 460 Val Asp Lys Lys Gln Pro Ala Asn His Ala Asp Ala Asn Lys
Ala Gln 465 470 475 480 Ala Leu Pro Glu Thr Gly Glu Glu Asn Pro Phe
Ile Gly Thr Thr Val 485 490 495 Phe Gly Gly Leu Ser Leu Ala Leu Gly
Ala Ala Leu Leu Ala Gly Arg 500 505 510 Arg Arg Glu Leu 515
6301PRTArtificial sequenceComparison SpA5 mutantSpA5ref(KKAA)
protein 6Gly Pro Leu Gly Ser Ala Gln Ala Gln Gln Asn Gln His Asp
Glu Ala 1 5 10 15 Lys Lys Asn Ala Phe Tyr Gln Val Leu Asn Met Pro
Asn Leu Asn Ala 20 25 30 Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu
Lys Ala Ala Pro Ser Gln 35 40 45 Ser Ala Asn Val Leu Gly Glu Ala
Gln Lys Leu Asn Asp Ser Gln Ala 50 55 60 Pro Lys Ala Asp Ala Lys
Lys Asn Asn Phe Asn Lys Asp Gln Gln Ser 65 70 75 80 Ala Phe Tyr Glu
Ile Leu Asn Met Pro Asn Leu Asn Glu Ala Gln Arg 85 90 95 Asn Gly
Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Thr Asn 100 105 110
Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 115
120 125 Asp Asn Asn Phe Asn Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile
Leu 130 135 140 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe
Ile Gln Ser 145 150 155 160 Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn
Leu Leu Ser Glu Ala Lys 165 170 175 Lys Leu Asn Glu Ser Gln Ala Pro
Lys Ala Asp Asn Lys Phe Asn Lys 180 185 190 Glu Lys Lys Asn Ala Phe
Tyr Glu Ile Leu His Leu Pro Asn Leu Asn 195 200 205 Glu Glu Gln Arg
Asn Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser 210 215 220 Gln Ser
Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 225 230 235
240 Ala Pro Lys Ala Asp Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala Phe
245 250 255 Tyr Glu Ile Leu His Leu Pro Asn Leu Thr Glu Glu Gln Arg
Asn Gly 260 265 270 Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser Val Ser
Lys Glu Ile Leu 275 280 285 Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln
Ala Pro Lys 290 295 300 7296PRTArtificial sequenceRecombinant SpA5
wild type protein (SpA5wt) 7Gly Pro Leu Gly Ser Ala Gln His Asp Glu
Ala Gln Gln Asn Ala Phe 1 5 10 15 Tyr Gln Val Leu Asn Met Pro Asn
Leu Asn Ala Asp Gln Arg Asn Gly 20 25 30 Phe Ile Gln Ser Leu Lys
Asp Asp Pro Ser Gln Ser Ala Asn Val Leu 35 40 45 Gly Glu Ala Gln
Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala 50 55 60 Gln Gln
Asn Lys Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile 65 70 75 80
Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln 85
90 95 Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu
Ala 100 105 110 Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn
Asn Phe Asn 115 120 125 Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu
Asn Met Pro Asn Leu 130 135 140 Asn Glu Glu Gln Arg Asn Gly Phe Ile
Gln Ser Leu Lys Asp Asp Pro 145 150 155 160 Ser Gln Ser Ala Asn Leu
Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser 165 170 175 Gln Ala Pro Lys
Ala Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala 180 185 190 Phe Tyr
Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn 195 200 205
Gly Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu 210
215 220 Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala
Asp 225 230 235 240 Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr
Glu Ile Leu His 245 250
255 Leu Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu
260 265 270 Lys Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
Lys Lys 275 280 285 Leu Asn Asp Ala Gln Ala Pro Lys 290 295
85PRTArtificial sequenceFrom pGEX-6P-2 vector series 8Gly Pro Leu
Gly Ser 1 5 9891DNAArtificial sequenceEncoding sequence of
recombinant SpA5(KKAA) protein 9gggcccctgg gatccgcgca acacgatgaa
gctaaaaaaa atgcttttta tcaagtgtta 60aatatgccta acttaaacgc tgatcaacgt
aatggtttta tccaaagcct taaagcagca 120ccaagccaaa gtgctaacgt
tttaggtgaa gctcaaaaac ttaatgactc tcaagctcca 180aaagctgatg
cgaaaaaaaa taagttcaac aaagatcaac aaagcgcctt ctatgaaatc
240ttgaacatgc ctaacttaaa cgaagagcaa cgcaatggtt tcattcaaag
tcttaaagca 300gcaccaagcc aaagcactaa cgttttaggt gaagctaaaa
aattaaacga atctcaagca 360ccgaaagctg acaacaattt caacaaagaa
aaaaaaaatg ctttctatga aatcttgaac 420atgcctaact tgaacgaaga
acaacgcaat ggtttcatcc aaagcttaaa agcagcacca 480agtcaaagtg
ctaacctttt agcagaagct aaaaagttaa atgaatctca agcaccgaaa
540gctgataaca aattcaacaa agaaaaaaaa aatgctttct atgaaatctt
acatttacct 600aacttaaatg aagaacaacg caatggtttc atccaaagct
taaaagcagc accaagccaa 660agcgctaacc ttttagcaga agctaaaaag
ctaaatgatg cacaagcacc aaaagctgac 720aacaaattca acaaagaaaa
aaaaaatgct ttctatgaaa ttttacattt acctaactta 780actgaagaac
aacgtaacgg cttcatccaa agccttaaag cagcaccttc agtgagcaaa
840gaaattttag cagaagctaa aaagctaaac gatgctcaag caccaaaata a
89110891DNAArtificial sequenceEncoding sequence of recombinant
SpA5(RRAA) protein 10gggcccctgg gatccgcgca acacgatgaa gctcgccgca
atgcttttta tcaagtgtta 60aatatgccta acttaaacgc tgatcaacgt aatggtttta
tccaaagcct taaagcagca 120ccaagccaaa gtgctaacgt tttaggtgaa
gctcaaaaac ttaatgactc tcaagctcca 180aaagctgatg cgcgccgcaa
taagttcaac aaagatcaac aaagcgcctt ctatgaaatc 240ttgaacatgc
ctaacttaaa cgaagagcaa cgcaatggtt tcattcaaag tcttaaagca
300gcaccaagcc aaagcactaa cgttttaggt gaagctaaaa aattaaacga
atctcaagca 360ccgaaagctg acaacaattt caacaaagaa cgccgcaatg
ctttctatga aatcttgaac 420atgcctaact tgaacgaaga acaacgcaat
ggtttcatcc aaagcttaaa agcagcacca 480agtcaaagtg ctaacctttt
agcagaagct aaaaagttaa atgaatctca agcaccgaaa 540gctgataaca
aattcaacaa agaacgccgc aatgctttct atgaaatctt acatttacct
600aacttaaatg aagaacaacg caatggtttc atccaaagct taaaagcagc
accaagccaa 660agcgctaacc ttttagcaga agctaaaaag ctaaatgatg
cacaagcacc aaaagctgac 720aacaaattca acaaagaacg ccgcaatgct
ttctatgaaa ttttacattt acctaactta 780actgaagaac aacgtaacgg
cttcatccaa agccttaaag cagcaccttc agtgagcaaa 840gaaattttag
cagaagctaa aaagctaaac gatgctcaag caccaaaata a 89111891DNAArtificial
sequenceEncoding sequence of recombinant SpA5(KKVV) protein
11gggcccctgg gatccgcgca acacgatgaa gctaaaaaaa atgcttttta tcaagtgtta
60aatatgccta acttaaacgc tgatcaacgt aatggtttta tccaaagcct taaagttgtt
120ccaagccaaa gtgctaacgt tttaggtgaa gctcaaaaac ttaatgactc
tcaagctcca 180aaagctgatg cgaaaaaaaa taagttcaac aaagatcaac
aaagcgcctt ctatgaaatc 240ttgaacatgc ctaacttaaa cgaagagcaa
cgcaatggtt tcattcaaag tcttaaagtt 300gttccaagcc aaagcactaa
cgttttaggt gaagctaaaa aattaaacga atctcaagca 360ccgaaagctg
acaacaattt caacaaagaa aaaaaaaatg ctttctatga aatcttgaac
420atgcctaact tgaacgaaga acaacgcaat ggtttcatcc aaagcttaaa
agttgttcca 480agtcaaagtg ctaacctttt agcagaagct aaaaagttaa
atgaatctca agcaccgaaa 540gctgataaca aattcaacaa agaaaaaaaa
aatgctttct atgaaatctt acatttacct 600aacttaaatg aagaacaacg
caatggtttc atccaaagct taaaagttgt tccaagccaa 660agcgctaacc
ttttagcaga agctaaaaag ctaaatgatg cacaagcacc aaaagctgac
720aacaaattca acaaagaaaa aaaaaatgct ttctatgaaa ttttacattt
acctaactta 780actgaagaac aacgtaacgg cttcatccaa agccttaaag
ttgttccttc agtgagcaaa 840gaaattttag cagaagctaa aaagctaaac
gatgctcaag caccaaaata a 89112891DNAArtificial sequenceEncoding
sequence of recombinant SpA5(RRVV) protein 12gggcccctgg gatccgcgca
acacgatgaa gctcgccgca atgcttttta tcaagtgtta 60aatatgccta acttaaacgc
tgatcaacgt aatggtttta tccaaagcct taaagttgtt 120ccaagccaaa
gtgctaacgt tttaggtgaa gctcaaaaac ttaatgactc tcaagctcca
180aaagctgatg cgcgccgcaa taagttcaac aaagatcaac aaagcgcctt
ctatgaaatc 240ttgaacatgc ctaacttaaa cgaagagcaa cgcaatggtt
tcattcaaag tcttaaagtt 300gttccaagcc aaagcactaa cgttttaggt
gaagctaaaa aattaaacga atctcaagca 360ccgaaagctg acaacaattt
caacaaagaa cgccgcaatg ctttctatga aatcttgaac 420atgcctaact
tgaacgaaga acaacgcaat ggtttcatcc aaagcttaaa agttgttcca
480agtcaaagtg ctaacctttt agcagaagct aaaaagttaa atgaatctca
agcaccgaaa 540gctgataaca aattcaacaa agaacgccgc aatgctttct
atgaaatctt acatttacct 600aacttaaatg aagaacaacg caatggtttc
atccaaagct taaaagttgt tccaagccaa 660agcgctaacc ttttagcaga
agctaaaaag ctaaatgatg cacaagcacc aaaagctgac 720aacaaattca
acaaagaacg ccgcaatgct ttctatgaaa ttttacattt acctaactta
780actgaagaac aacgtaacgg cttcatccaa agccttaaag ttgttccttc
agtgagcaaa 840gaaattttag cagaagctaa aaagctaaac gatgctcaag
caccaaaata a 89113290PRTArtificial sequenceRecombinant MntC protein
13Gly Pro Leu Gly Ser Ser Ser Asp Lys Ser Asn Gly Lys Leu Lys Val 1
5 10 15 Val Thr Thr Asn Ser Ile Leu Tyr Asp Met Ala Lys Asn Val Gly
Gly 20 25 30 Asp Asn Val Asp Ile His Ser Ile Val Pro Val Gly Gln
Asp Pro His 35 40 45 Glu Tyr Glu Val Lys Pro Lys Asp Ile Lys Lys
Leu Thr Asp Ala Asp 50 55 60 Val Ile Leu Tyr Asn Gly Leu Asn Leu
Glu Thr Gly Asn Gly Trp Phe 65 70 75 80 Glu Lys Ala Leu Glu Gln Ala
Gly Lys Ser Leu Lys Asp Lys Lys Val 85 90 95 Ile Ala Val Ser Lys
Asp Val Lys Pro Ile Tyr Leu Asn Gly Glu Glu 100 105 110 Gly Asn Lys
Asp Lys Gln Asp Pro His Ala Trp Leu Ser Leu Asp Asn 115 120 125 Gly
Ile Lys Tyr Val Lys Thr Ile Gln Gln Thr Phe Ile Asp Asn Asp 130 135
140 Lys Lys His Lys Ala Asp Tyr Glu Lys Gln Gly Asn Lys Tyr Ile Ala
145 150 155 160 Gln Leu Glu Lys Leu Asn Asn Asp Ser Lys Asp Lys Phe
Asn Asp Ile 165 170 175 Pro Lys Glu Gln Arg Ala Met Ile Thr Ser Glu
Gly Ala Phe Lys Tyr 180 185 190 Phe Ser Lys Gln Tyr Gly Ile Thr Pro
Gly Tyr Ile Trp Glu Ile Asn 195 200 205 Thr Glu Lys Gln Gly Thr Pro
Glu Gln Met Arg Gln Ala Ile Glu Phe 210 215 220 Val Lys Lys His Lys
Leu Lys His Leu Leu Val Glu Thr Ser Val Asp 225 230 235 240 Lys Lys
Ala Met Glu Ser Leu Ser Glu Glu Thr Lys Lys Asp Ile Phe 245 250 255
Gly Glu Val Tyr Thr Asp Ser Ile Gly Lys Glu Gly Thr Lys Gly Asp 260
265 270 Ser Tyr Tyr Lys Met Met Lys Ser Asn Ile Glu Thr Val His Gly
Ser 275 280 285 Met Lys 290 14240PRTArtificial sequenceRecombinant
mSEB protein 14Met Glu Ser Gln Pro Asp Pro Lys Pro Asp Glu Leu His
Lys Ser Ser 1 5 10 15 Lys Phe Thr Gly Leu Met Glu Asn Met Lys Val
Leu Tyr Asp Asp Asn 20 25 30 His Val Ser Ala Ile Asn Val Lys Ser
Ile Asp Gln Phe Arg Tyr Phe 35 40 45 Asp Leu Ile Tyr Ser Ile Lys
Asp Thr Lys Leu Gly Asn Tyr Asp Asn 50 55 60 Val Arg Val Glu Phe
Lys Asn Lys Asp Leu Ala Asp Lys Tyr Lys Asp 65 70 75 80 Lys Tyr Val
Asp Val Phe Gly Ala Asn Ala Tyr Tyr Gln Cys Ala Phe 85 90 95 Ser
Lys Lys Thr Asn Asp Ile Asn Ser His Gln Thr Asp Lys Arg Lys 100 105
110 Thr Cys Met Tyr Gly Gly Val Thr Glu His Asn Gly Asn Gln Leu Asp
115 120 125 Lys Tyr Arg Ser Ile Thr Val Arg Val Phe Glu Asp Gly Lys
Asn Leu 130 135 140 Leu Ser Phe Asp Val Gln Thr Asn Lys Lys Lys Val
Thr Ala Gln Glu 145 150 155 160 Leu Asp Tyr Leu Thr Arg His Tyr Leu
Val Lys Asn Lys Lys Leu Tyr 165 170 175 Glu Phe Asn Asn Ser Pro Tyr
Glu Thr Gly Tyr Ile Lys Phe Ile Glu 180 185 190 Asn Glu Asn Ser Phe
Trp Tyr Asp Met Met Pro Ala Pro Gly Asp Lys 195 200 205 Phe Asp Gln
Ser Lys Tyr Leu Met Met Tyr Asn Asp Asn Lys Met Val 210 215 220 Asp
Ser Lys Asp Val Lys Ile Glu Val Tyr Leu Thr Thr Lys Lys Lys 225 230
235 240 15424PRTArtificial sequenceRecombinant HI protein 15Gly Pro
Leu Gly Ser Met Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly 1 5 10 15
Thr Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val 20
25 30 Thr Tyr Asp Lys Glu Asn Gly Met Leu Lys Lys Val Phe Tyr Ser
Phe 35 40 45 Ile Asp Asp Lys Asn His Asn Lys Lys Ile Leu Val Ile
Arg Thr Lys 50 55 60 Gly Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser
Glu Glu Gly Ala Asn 65 70 75 80 Lys Ser Gly Leu Ala Trp Pro Ser Ala
Phe Lys Val Gln Leu Gln Leu 85 90 95 Pro Asp Asn Glu Val Ala Gln
Ile Ser Asp Tyr Tyr Pro Arg Asn Ser 100 105 110 Ile Asp Thr Lys Glu
Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly 115 120 125 Asn Val Thr
Gly Asp Asp Ser Gly Lys Ile Gly Gly Leu Ile Gly Ala 130 135 140 Asn
Val Ser Ile Gly His Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys 145 150
155 160 Thr Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val
Ile 165 170 175 Phe Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr Asp
Arg Asp Ser 180 185 190 Trp Asn Pro Val Tyr Gly Asn Gln Leu Phe Met
Lys Thr Arg Asn Gly 195 200 205 Ser Met Lys Ala Ala Glu Asn Phe Leu
Asp Pro Asn Lys Ala Ser Ser 210 215 220 Leu Leu Ser Ser Gly Phe Ser
Pro Asp Phe Ala Thr Val Ile Thr Met 225 230 235 240 Asp Arg Lys Ala
Thr Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu 245 250 255 Arg Val
Arg Asp Asp Tyr Gln Leu His Trp Thr Ser Thr Asn Trp Lys 260 265 270
Gly Thr Asn Thr Lys Asp Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr 275
280 285 Lys Ile Asp Trp Glu Lys Glu Glu Met Thr Asn Gly Gly Gly Gly
Ser 290 295 300 Lys Met Thr Asp Leu Gln Asp Thr Lys Tyr Val Val Tyr
Glu Ser Val 305 310 315 320 Glu Asn Asn Glu Ser Met Met Asp Ala Phe
Val Lys His Pro Ile Lys 325 330 335 Thr Gly Met Leu Asn Gly Lys Lys
Tyr Met Val Met Glu Thr Thr Asn 340 345 350 Asp Asp Tyr Trp Lys Asp
Phe Met Val Glu Gly Gln Arg Val Arg Thr 355 360 365 Ile Ser Lys Asp
Ala Lys Asn Asn Thr Arg Thr Ile Ile Phe Pro Tyr 370 375 380 Val Glu
Gly Lys Thr Leu Tyr Asp Ala Ile Val Lys Val His Val Lys 385 390 395
400 Thr Ile Asp Tyr Asp Gly Gln Tyr His Val Arg Ile Val Asp Lys Glu
405 410 415 Ala Phe Thr Lys Ala Asn Leu Glu 420 1615DNAArtificial
sequenceEncoding sequence of amino acid fragment GPLGS from
pGEX-6P-2 16gggcccctgg gatcc 15
* * * * *
References